Aims

cAMP response element binding protein (CREB1) is involved in the progression of osteoarthritis (OA). However, available findings about the role of CREB1 in OA are inconsistent. 666-15 is a potent and selective CREB1 inhibitor, but its role in OA is unclear. This study aimed to investigate the precise role of CREB1 in OA, and whether 666-15 exerts an anti-OA effect.

Aims

To investigate the optimal thresholds and diagnostic efficacy of commonly used serological and synovial fluid detection indexes for diagnosing periprosthetic joint infection (PJI) in patients who have rheumatoid arthritis (RA).

To evaluate the therapeutic effect of Pulsed Electromagnetic Field (PEMF) in the treatment of meniscal tears in the avascular region.

Seventy-two twelve-week-old male Sprague-Dawley rats with full-thickness longitudinal medial meniscal tears in the avascular region were divided into 3 groups: control group (G_con), treated with classic signal PEMF (G_classic), and high slew rate signal PEMF(G_HSR). The HSR signal has the same pulse and burst frequencies as the classic signal, but with a higher slew rate. Macroscopic observation and histological analysis of the meniscus and articular cartilage were performed to evaluate the meniscal healing and progressions of osteoarthritis. The synovium was harvested for histological and immunofluorescent analysis to assess the intra-articular inflammation. The meniscal healing, articular cartilage degeneration, and synovitis were quantitatively evaluated according to their respective scoring system.

Dramatic degenerative changes of the meniscus and articular cartilage were noticed during gross observation and histological evaluation in the control group at 8 weeks. However, the menisci in the two treatment groups were restored to normal morphology with a smooth surface and shiny white color. Particularly, the HSR signal remarkably enhanced the fibrochondrogenesis and accelerated the remodeling process of the regenerated tissue. The meniscal healing scores of PEMF treatment groups were significantly higher than those in the control group at 8 weeks. Specifically, the HSR signal showed a significantly higher meniscal repair score than the classic signal at week 8 (P < .01). The degeneration score (G_con versus G_classic: P < .0001; Gcon versus G_HSR: P < .0001) and synovitis score (G_con versus Gclassic: P < .0001; G_con versus G_HSR: P = .0002) of the control groups were significantly higher than those in the two treatment groups.

PEMF promoted the healing of meniscal tears in the avascular region and restored the injured meniscus to its structural integrity in a rat model. Compared to the classic signal, the HSR signal showed the increased capability to promote fibrocartilaginous tissue formation and modulate the inflammatory environment and therefore protected the knee joint from post-traumatic osteoarthritis development.

Critical size bone defects are frequently caused by accidental trauma, oncologic surgery, and infection. Distraction osteogenesis (DO) is a useful technique to promote the repair of critical size bone defects. However, DO is usually a lengthy treatment, therefore accompanied with increased risks of complications such as infections and delayed union.

Herein, we developed an innovative intramedullary biodegradable magnesium (Mg) nail to accelerate bone regeneration in critical size bone defect repair during DO.

We observed that Mg nail induced almost 4-fold increase of new bone formation and over 5-fold of new vessel formation at 2 weeks after distraction. Mg nail upregulated the expression of calcitonin gene-related peptide (CGRP) in the new bone as compared with the DO alone group. We further revealed that blockade of the sensory nerve by overdose capsaicin blunted Mg nail enhanced critical size bone defect repair during the DO process. Moreover, inhibitors/antagonist of CGRP receptor, FAK, and VEGF receptor blocked the Mg nail stimulated vessel and bone formation.

In summary, we revealed, for the first time, a CGRP-FAK-VEGF signaling axis linking sensory nerve and endothelial cells, which may be the main mechanism underlying Mg-enhanced critical size bone defect repair when combined with DO, suggesting a great potential of Mg implants in reducing DO treatment time for clinical applications.

Based on Ilizarov's law of tension-stress principle, distraction histogenesis technique has been widely applied in orthopaedic surgery for decades. Derived from this technique, cranial bone transport technique was mainly used for treating cranial deformities and calvarial defects. Recent studies reported that there are dense short vascular connections between skull marrow and meninges for immune cells trafficking, highlighting complex and tight association between skull and brain. Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia without effective therapy. Meningeal lymphatics have been recognized as an important mediator in neurological diseases. The augmentation of meningeal lymphatic drainage might be a promising therapeutic target for AD. Our proof-of-concept study has indicated that cranial bone transport can promote ischemic stroke recovery via modulating meningeal lymphatic drainage function, providing a rationale for treating AD using cranial bone maneuver (CBM). This study aims to investigate the effects of CBM on AD and to further explore the potential mechanisms.

Transgenic 5xFAD mice model was used in this study. After osteotomy, a bone flap was used to perform CBM without damaging the dura. Open filed test, novel object recognition test and Barn's maze test were used to evaluate neurological functions of 5xFAD mice after CBM treatment. Congo red and immunofluorescence staining were used to evaluate amyloid depositions and Aβ plaques in different brain regions. Lymphangiogenesis and the level of VEGF-C were examined after CBM treatment. OVA-A647 was intra-cisterna-magna injected to evaluate meningeal lymphatic drainage function after CBM treatment.

CBM significantly improved memory functions and reduced amyloid depositions and Aβ plaques in the hippocampus of 5xFAD mice. A significant increase of meningeal lymphatic vessels in superior sagittal sinus and transverse sinus, and the upregulation of VEGF-C in meninges were observed in 5xFAD mice treated with CBM. Moreover, CBM remarkably enhanced meningeal lymphatic drainage function in 5xFAD mice (n=5-16 mice/group for all studies).

CBM may promote meningeal lymphangiogenesis and lymphatic drainage function through VEGF-C-VEGFR3 pathway, and further reduce amyloid depositions and Aβ plaques and alleviate memory deficits in AD.

Treatment for delayed wound healing resulting from peripheral vascular diseases and diabetic foot ulcers remain a challenge. A novel surgical technique named Tibial Cortex Transverse Transport has been developed for treating peripheral ischaemia, with encouraging clinical effects. However, its underlying mechanisms remain unclear. In present study, we aimed to explore the wound healing effects after undergoing this novel technique via multiple ways.

A novel rat model of Tibial Cortex Transverse Transport was established with a designed external fixator and effects on wound healing were investigated. All rats were randomized into 3 groups, with 12 rats per group: sham group (negative control), fixator group (positive control) and Tibial Cortex Transverse Transport group. Laser speckle perfusion imaging, vessel perfusion, histology and immunohistochemistry were used to evaluate the wound healing processes.

Gross and histological examinations showed that Tibial Cortex Transverse Transport technique accelerated wound closure and enhanced the quality of the newly formed skin tissues. In Tibial Cortex Transverse Transport group, HE staining demonstrated a better epidermis and dermis recovery, while immune-histochemical staining showed that Tibial Cortex Transverse Transport technique promoted local collagen deposition. Tibial Cortex Transverse Transport technique also benefited to angiogenesis and immunomodulation. In Tibial Cortex Transverse Transport group, blood flow in the wound area was higher than that ofother groups according to laser speckle imaging with more blood vessels observed. Enhanced neovascularization was seen in the Tibial Cortex Transverse Transport group with double immune-labelling of CD31 and α-SMA. The M2 macrophages at the wound site in the Tibial Cortex Transverse Transport group was also increased.

Tibial cortex transverse transport technique accelerated wound healing through enhanced angiogenesis and immunomodulation.

Osteoarthritis (OA) is a highly prevalent degenerative joint disorder characterized by joint pain and physical disability. Aberrant subchondral bone induces pathological changes and is a major source of pain in OA. In the subchondral bone, which is highly innervated, nerves have dual roles in pain sensation and bone homeostasis regulation. The interaction between peripheral nerves and target cells in the subchondral bone, and the interplay between the sensory and sympathetic nervous systems, allow peripheral nerves to regulate subchondral bone homeostasis. Alterations in peripheral innervation and local transmitters are closely related to changes in nociception and subchondral bone homeostasis, and affect the progression of OA. Recent literature has substantially expanded our understanding of the physiological and pathological distribution and function of specific subtypes of neurones in bone. This review summarizes the types and distribution of nerves detected in the tibial subchondral bone, their cellular and molecular interactions with bone cells that regulate subchondral bone homeostasis, and their role in OA pain. A comprehensive understanding and further investigation of the functions of peripheral innervation in the subchondral bone will help to develop novel therapeutic approaches to effectively prevent OA, and alleviate OA pain.

Cite this article: Bone Joint Res 2022;11(7):439–452.

Aims

Periprosthetic joint infections (PJIs) with prior multiple failed surgery for reinfection represent a huge challenge for surgeons because of poor vascular supply and biofilm formation. This study aims to determine the results of single-stage revision using intra-articular antibiotic infusion in treating this condition.

Aims

Treatment for delayed wound healing resulting from peripheral vascular diseases and diabetic foot ulcers remains a challenge. A novel surgical technique named ‘tibial cortex transverse transport’ (TTT) has been developed for treating peripheral ischaemia, with encouraging clinical effects. However, its underlying mechanisms remain unclear. In the present study, we explored the potential biological mechanisms of TTT surgery using various techniques in a rat TTT animal model.

Aims

Distraction osteogenesis (DO) is a useful orthopaedic procedure employed to lengthen and reshape bones by stimulating bone formation through controlled slow stretching force. Despite its promising applications, difficulties are still encountered. Our previous study demonstrated that pulsed electromagnetic field (PEMF) treatment significantly enhances bone mineralization and neovascularization, suggesting its potential application. The current study compared a new, high slew rate (HSR) PEMF signal, with different treatment durations, with the standard Food and Drug Administration (FDA)-approved signal, to determine if HSR PEMF is a better alternative for bone formation augmentation.

Aims

Ageing-related incompetence becomes a major hurdle for the clinical translation of adult stem cells in the treatment of osteoarthritis (OA). This study aims to investigate the effect of stepwise preconditioning on cellular behaviours in human mesenchymal stem cells (hMSCs) from ageing patients, and to verify their therapeutic effect in an OA animal model.

Aims

The aim of this study was to systematically compare the safety and accuracy of robot-assisted (RA) technique with conventional freehand with/without fluoroscopy-assisted (CT) pedicle screw insertion for spine disease.

Introduction

Medial pivoting motion of the knee has been widely assumed in total knee arthroplasty (TKA) research, but was not consistently observed in recent studies of in vivo knee motion. This study investigated the in vivo motion characters of the knee by analyzing the axial tibial rotation and tibiofemoral articular contact motion during a weightbearing flexion and a treadmill gait.

Aims

In the absence of an identified organism, single-stage revision is contraindicated in prosthetic joint infection (PJI). However, no studies have examined the use of intra-articular antibiotics in combination with single-stage revision in these cases. In this study, we present the results of single-stage revision using intra-articular antibiotic infusion for treating culture-negative (CN) PJI.

Objectives

Activation of the leptin pathway is closely correlated with human knee cartilage degeneration. However, the role of the long form of the leptin receptor (Ob-Rb) in cartilage degeneration needs further study. The aim of this study was to determine the effect of increasing the expression of Ob-Rb on chondrocytes using a lentiviral vector containing Ob-Rb.

Aims

Single-stage revision is not widely pursued due to restrictive inclusion criteria. In this study, we evaluated the results of single-stage revision of chronically infected total hip arthroplasty (THA) using broad inclusion criteria and cementless implants.

Distraction histogenesis (DH) techniques have been widely accepted and practiced in orthopaedics, traumatology, and craniofacial surgery over the last two decades. Using DH methods, many previously untreatable conditions have been successfully managed with outstanding clinical outcomes. The biological mechanisms underlying DH have been studied and the tension-stress principles of tissue regeneration are attributed to upregulated gene expression, enhanced cell proliferation, angiogenesis and tissue remodelling and endogenous stem cell mobilization. The new methods of enhancing bone consolidation in DH are proposed and need further clinical studies. The novel applications of DH have now been extended for the treatment of vascular diseases, cranial defect (with neuronal disorders), hip and spinal deformity corrections and soft-tissue defects in addition to various bone defects and deformities. There are more surprises and novel mechanisms yet to be discovered for these novel applications of DH.

Objectives

The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known.

Objectives

As one of the heat-stable enterotoxins, Staphylococcal enterotoxin C2 (SEC2) is synthesized by Staphylococcus aureus, which has been proved to inhibit the growth of tumour cells, and is used as an antitumour agent in cancer immunotherapy. Although SEC2 has been reported to promote osteogenic differentiation of human mesenchymal stem cells (MSCs), the in vivo function of SCE2 in animal model remains elusive. The aim of this study was to further elucidate the in vivo effect of SCE2 on fracture healing.

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mass and deterioration of bone microarchitecture, which results in increased bone fragility and fracture risk. Casein kinase 2-interacting protein-1 (CKIP-1) is a protein that plays an important role in regulation of bone formation. The effect of CKIP-1 on bone formation is mainly mediated through negative regulation of the bone morphogenetic protein pathway. In addition, CKIP-1 has an important role in the progression of osteoporosis. This review provides a summary of the recent studies on the role of CKIP-1 in osteoporosis development and treatment.

Cite this article: X. Peng, X. Wu, J. Zhang, G. Zhang, G. Li, X. Pan. The role of CKIP-1 in osteoporosis development and treatment. Bone Joint Res 2018;7:173–178. DOI: 10.1302/2046-3758.72.BJR-2017-0172.R1.

Objectives

This study looked to analyse the expression levels of microRNA-140-3p and microRNA-140-5p in synovial fluid, and their correlations to the severity of disease regarding knee osteoarthritis (OA).

Objectives

The length of the tourniquet time during total knee arthroplasty (TKA) is related to the incidence of post-operative deep vein thrombosis (DVT). Our aim in this study was to investigate the effect of the early release of the tourniquet on the incidence of DVT in patients undergoing TKA.

Objectives

Osteoporosis has become an increasing concern for older people as it may potentially lead to osteoporotic fractures. This study is designed to assess the efficacy and safety of ten therapies for post-menopausal women using network meta-analysis.

Objectives

Distraction osteogenesis (DO) mobilises bone regenerative potential and avoids the complications of other treatments such as bone graft. The major disadvantage of DO is the length of time required for bone consolidation. Mesenchymal stem cells (MSCs) have been used to promote bone formation with some good results.

Introduction

An equal knee joint height during flexion and extension is of critical importance in optimizing soft-tissue balancing following total knee arthroplasty (TKA). However, there is a paucity of data regarding the in-vivo knee joint height behavior. This study evaluated in-vivo heights and anterior-posterior (AP) translations of the medial and lateral femoral condyles before and after a cruciate-retaining (CR)-TKA using two flexion axes: surgical transepicondylar axis (sTEA) and geometric center axis (GCA).

INTRODUCTION

In native knees anterior cruciate ligament (ACL) and asymmetric shape of the tibial articular surface with a convex lateral plateau are responsible for differential medial and lateral femoral rollback. Contemporary ACL retaining total knee arthroplasty (TKA) improves knee function over ACL sacrificing (CR) TKA; however, these implants do not restore the asymmetric tibial articular geometry. This may explain why ACL retention addresses paradoxical anterior sliding seen in CR TKA, but does not fully restore medial pivot motion. To address this, an ACL retaining biomimetic implant, was designed by moving the femoral component through healthy in vivo kinematics obtained from bi-planar fluoroscopy and sequentially removing material from a tibial template. We hypothesized that the biomimetic articular surface together with ACL preservation would better restore activity dependent kinematics of normal knees, than ACL retention alone.

Combined acetabular and femoral anteversion (CA) of the hip following total hip arthroplasty (THA) is critical to the hip function and longevity of the components. However, no study has been reported on the accuracy in restoration of CA of the hip after operation using robotic assistance and conventional free-hand techniques. The purpose of this study was to evaluate if using robotic assistance in THA can better restore native CA than a free-hand technique.

Twenty three unilateral THA patients participated in this study. Twelve of them underwent a robotic-arm assisted THA (RIO® Robotic Arm Interactive Orthopedic System, Stryker Mako., Fort Lauderdale, FL, USA) and eleven received a free-hand THA. Subject specific 3D models of both implanted and non-implanted hips were reconstructed using post-operative CT scans. The anteversion and inclination of the native acetabulum and implanted cup were measured and compared. To determine the differences of the femoral anteversion between sides, the non-implanted native femur was mirrored and aligned with the remaining femur of the implanted side using an iterative closest point algorithm. The angle between the native femoral neck axis and the prosthesis neck axis in transverse plane was measured as the change in femoral anteversion following THA. The sum of the changes of the acetabular and femoral anteversion was defined as the change of CA after THA. A Wilcoxon signed rank test was performed to test if the anteversion of the navigation and free-hand THAs were different from the contralateral native hips (α = 0.05).

The acetabular anteversion were 22.0°±7.4°, 35.9°±6.5° and 32.6°±22.6° for the native hips, robotic assisted THAs and free-hand THAs, respectively, and the corresponding values of the acetabular inclinations were 52.0°±2.9°, 35.4°±4.4° and 43.1°±7.1°. The acetabular anteversion was increased by 12.2°±11.1° (p=0.005) and 12.5°±20.0° (p=0.102) for the robotic assisted and the free-hand THAs. The femoral anteversion was increased by 6.3°±10.5° (p=0.077) and 11.0°±13.4° (p=0.014) for the robotic assisted and free-hand THAs, respectively. The CA were significantly increased by 18.5°±11.7° (p<0.001) and 23.5°±26.5° (p=0.019) for the robotic assisted and the free-hand THAs. The changes of the CA of the free-hand THAs varied in a larger range than those of the robotic assisted THAs.

This study is the first to evaluate the changes in acetabular and femoral anteversions of the hips after robotic assisted and free-hand THAs using the contralateral native hip as a control. The results demonstrate that both the navigation and free-hand THAs significantly increased the CA compared to the contralateral native hips, but the changes of the robotic assisted THAs (18.5°±11.7°) were smaller and varied less than those of the free-hand THAs (23.5°±26.5°). These data suggest that the robotic assisted THA can better restore the native hip CAs with higher repeatability than the free-hand technique. Further studies are needed to investigate the effects of the hip anteversion changes on the in-vivo function of the hip and the long-term outcomes in THA patients.

Summary Statement

Umbilical cord derived stem cell secretion could enhance the osteogenic differentiation of human bone marrow stem cells. It may promote bone, cartilage and tendon regeneration in rat models, but the effect was not significant up to now.

INTRODUCTION

Contemporary PCL sacrificing Total Knee Arthroplasty (TKA) implants (CS) consist of symmetric medial and lateral tibial articular surfaces with high anterior lips designed to substitute for the stability of the native PCL. However, designs vary significantly across implant systems in the level of anteroposterior constraint provided. Therefore, the goal of this study was to investigate kinematics of two CS designs with substantially different constraint levels. The hypothesis was that dynamic knee simulations could show the effect of implant constraint on kinematics of CS implants.

Introduction:

While kinematic abnormalities of contemporary TKA implants have been well established, a solution has not yet been achieved. We hypothesized that contemporary TKA implants are not compatible with normal soft-tissue function and normal knee motion. We propose a novel technique for reverse engineering advanced implant articular surfaces (biomimetic surface), by using accurate 3D kinematics of normal knees. This technique accounts for surgical placement of the implants, and allows design of tibial and femoral articular surfaces in conjunction.

Due to technology advancement, many studies have reported on in-vivo human knee kinematics recently (Dannis, 2005; Moro-oka, 2008; Tashman, 2003; Koo, 2008). This abstract summarized the joint kinematics during three motions usually seen in our daily living, i.e. gait, step-up (stair ascending) and single-legged lunge that was measured using a combined dual-fluoroscopic imaging system and MRI based modeling technique (Li, 2008). Cartilage contacts or condylar motion using transepicondylar axis (TEA)/geometric center axis (GCA) were used to describe the motion characters of the knee during these motions.

In the treadmill gait, the movement of the medial femoral condyle along the anteroposterior direction was significantly greater than that of the lateral femoral condyle during the stance phase using either TEA (9.7 ± 0.7 mm vs. 4.0 ± 1.7 mm, respectively; p < 0.01; Fig. 1A) or GCA (17.4 ± 2.0 mm vs. 7.4 ± 6.1 mm, respectively; p < 0.01; Fig. 1B). A “lateral-pivoting” of the knee was observed (Kozanek, 2009).

In the step-up motion, both medial and lateral contact points moved anteriorly on the tibial articular surfaces along the step-up motion path. The contact points on the medial and lateral tibial plateau moved anteriorly (13.5 ± 3.2 mm vs. 10.7 ± 5.0 mm, respectively; p > 0.05; Fig. 2A) with knee extension. Using the TEA (Fig. 2B), the femoral condylar motions presented a similar pattern as the contact points; nonetheless, using the GCA (Fig. 2C), the femoral condylar motion pattern was dramatically different. The medial condyle moved anteriorly, while the lateral condyle shifted posteriorly. However, none of them showed a significant pivoting phenomenon (Li, 2013).

In the single-legged lunge, both medial and lateral contact points moved similarly before 120° of knee flexion, but the lateral contact moved posteriorly and significantly more than the medial compartment in high flexion (1.9 ± 2.1 mm vs. 4.8 ± 2 mm, respectively; p < 0.05). The single-legged lunge didn't show a single motion pattern (Fig. 3) (Qi, 2013).

These data provide baseline knowledge for the understanding of normal physiological function of the knee during gait, step-up and lunge activities. The findings of these studies demonstrated that knee joint kinematics is activity-dependent and indicated that the knee joint motions could not be described using a single motion character such as “medial-pivoting” that has recently been popularized in total knee arthroplasty design areas.

Introduction:

Contemporary Posterior Cruciate Ligament (PCL) retaining TKA implants (CR) are associated with well-known kinematic deficits, such as absence of medial pivot motion, paradoxical anterior femoral sliding, and posterior femoral subluxation at full extension. The hypothesis of this study was that a biomimetic implant, reverse engineered by using healthy knee kinematics to carve the tibial articular surface, could restore normal kinematic patterns of the knee.

INTRODUCTION

ACL retaining (BCR) Total Knee Arthroplasty (TKA) provides more normal kinematics than ACL sacrificing (CR) TKA. However, in the native knee the ACL and the asymmetric shape of the tibial articular surface with a convex lateral plateau are responsible for the differential medial/lateral femoral rollback (medial pivot). Therefore, the hypothesis of this study was that an asymmetric biomimetic articular surface together with ACL preservation would better restore native knee kinematics than retention of the ACL alone. Normal knee kinematics from bi-planar fluoroscopy was used to reverse engineer the tibial articular surface of the biomimetic implant. This was achieved by moving the femoral component through the healthy knee kinematics and removing material from a tibial template.

Ten percent of fractures end in delayed or non-union. NSAIDs have been linked to an inhibitory action on fracture repair for three decades yet the mechanism of action remains to be elucidated. Cancer research has identified that NSAIDs impede cell proliferation by inhibiting angiogenesis. It is proposed that a similar mechanism occurs in the induction of NSAID induced non-union. We have investigated this hypothesis in a randomised placebo control trial of the NSAID rofecoxib using a murine femoral fracture.

All animals had an open femoral fracture treated using an external fixator. Outcomes measures included x-ray, histology and biomechanical testing, with laser Doppler used to assess blood flow across the fracture gap.

Radiology showed similar healing patterns in both groups; however, at the later stages (day 32) the NSAID group had significantly poorer healing. Histological analysis showed that controls healed quicker (days 24 and 32), with more callus (day 8) and less fibrous tissue (Day 32). Biomechanical testing showed controls were stronger at day 32. Both groups exhibited a similar pattern of blood flow; however NSAIDs exhibited a lower median flow from day 4 onwards (significant at days 4, 16 and 24).

Positive correlations were demonstrated between both histological and radiographic assessments of healing, with increasing blood flow. NSAID animals exhibited lower flows and poorer healing by all outcomes. Regression analysis demonstrates, however, that the negative effect of NSAIDs on fracture repair is independent of its inhibitory action on blood flow.

COX-2 inhibitors are marketed as having cleaner side effect profiles and are widely used in trauma patients. Following development of a novel method of analysing functional vascularity across a fracture gap, we have demonstrated that the COX-2 inhibitor rofecoxib has a significant negative effect on blood flow at the fracture gap alongside inhibiting fracture repair.

Introduction: MSCs were demonstrated to exist within peripheral blood (PB) of several mammalian species including human, guinea pig, mice, rat, and rabbit. We have found increased numbers of circulating MSCs in human peripheral blood after fracture and in patients with cancers. We have also compared the difference between circulating MSCs and bone marrow MSCs and evaluated their potential clinical applications in tissue engineering and cell therapy.

Methods and findings: Using culture conditions similar to those defined for bone marrow derived mesenchymal stromal cells (BMMSCs), we have isolated and expanded multi-colony and single colony derived PBMSCs strains from the GFP transgenic rats. Aspects of molecular, cellular and developmental properties of this poorly characterized peripheral blood subpopulation were examined. PBMSCs share some common phenotypic characteristics with BMMSCs, but are distinguishable in gene expression profile by cDNA microarray analysis, with 84 up-regulated and 83 down-regulated genes (> 2 fold, E-B/B-E> 100, P< 0.05). Most of these genes are related to cell proliferation, differentiation, cyto-skeleton, and calcium/iron homeostasis. Differentially expressed genes with fold change ≥10 were further confirmed with quantitative real time RT-PCR, and these genes are: retinol-binding protein 1 (CRBP1), cadherin 2, bone morphogenetic protein 6 (BMP6), SRY-box containing gene 11 (Sox11), the aquaporin 1 (AQP1), and so on, and they can be potential targets for further investigations. We have demonstrated that single colony derived PBMSCs strains possess extensive proliferation and multipotent differentiation potentials into osteoblasts, adipocytes, chondrocytes, endothelial cells and neuronal cells. In terms of potential clinical implications of PBMSCs, we have demonstrated that allogenic PB-MSCs enhance bone regeneration in rabbit ulna critical-sized bone defect model. We also demonstrated that BM-MSCs can be recruited towards to the sites of bone fracture and participate fracture healing. We are now working on using MSCs as a gene delivery vehicle for management of would healing or cancer therapy, and ways of enhancing the homing and recruitment MSCs towards to specific sites after their systemic delivery.

Conclusion: Taken the above data together, PB-MSCs may be a new cell source for cell therapy, tissue engineering and gene therapy strategies.

Introduction: This study tested the hypothesis that the use of biomaterials in distraction osteogenesis (DO) would reduce the treatment time and enhance bone formation in bone defect management.

Methods: A 1.0cm tibial shaft was removed in the left tibia of 36 rabbits. Rabbits were divided into three groups: Group A, the defect gap was reduced with the tibia shortened for 1.0-cm; Group B, the defect gap was filled with 1.0-cm porous hydroxyapatite/tri-calcium phosphates cylindrical block (HA/TCP block, diameter 0.5-cm); Group C, The 1.0-cm defect gap was reduced 0.5cm and the remaining 0.5-cm defect gap was filled with 0.5-cm HA/TCP block. The tibia was then fixed with unilateral lengthener; for groups A and C, lengthening started 7 days after surgery at a rate of 1.0 mm/day, in two steps. Group A received lengthening for 10 days and Group C for 5 days, there was no lengthening for Group B. All animals were terminated at day 37 following surgery. The excised bone specimens were subject to micro-CT, mechanical testing and histological examinations.

Results: Bone mineral density and content and tissue mineral density and content, as well as the mechanical properties of the regenerates were significantly higher in Group C compared to Groups A and B. Micro CT and histological examinations also confirmed that the regenerates in Group C had most advanced bone formation, consolidation and remodeling compared to other groups.

Conclusion: The combined use of biomaterials and DO technique can reduce the treatment time and enhance bone consolidation in bone defect management.

Thrombin related peptide (TP 508) is a 23 amino-acid synthetic peptide that mimics a portion of the receptor-binding domain of the human thrombin molecule.

Thrombin triggers both proteolytic activated receptors and non proteolytic activated receptors to bring about a mixture of responses ranging from tissue breakdown and clot formation, to new vessel formation and tissue repair. TP 508 stimulates only the non proteolytic activated receptors, and this initiates repair and angiogenesis but not clot formation or tissue breakdown Previous studies have shown that TP508 can stimulate repair in the dermal and musculoskeletal tissues by promoting angiogenesis and enhancing the proliferation and migration of cells.

High energy fractures are associated with a delay in healing. We hypothesized that high energy fracture healing would be improved with the use of TP508, and that the dose and site of application would have importance.

Methods: 80 CD 1 Mice were randomised into four groups; all underwent a high energy quadriceps muscle crush and a femoral fracture on the left hind limb. In each case the fracture was reduced and held with an external fixator. At the time of operation Group I received a dose of 100ìg TP 508 into the fracture, Group II 100ìg into the surrounding damaged soft tissue, Group III a dose of 10ìg into the fracture, and group IV (the control group) received PBS carrier into the fracture.

24 animals were sacrificed on day 21 and the remaining 56 mice on day 35. Of the 35 day old animals 8 in each group had both femora harvested and the biomechanical properties were tested using the 3-point bending technique. Specimens from the 21 day old animals and remaining 35 day old animals were used for histological analysis.

All 80 animals had digital radiographs taken each week. Using image analysis software five pixel density graphs were generated across each fracture gap. A validated semi quantitative analysis was used to score each graph and the total accumulated for each radiograph. The width of the fracture calus was measured and expressed as a ratio of the femur diameter.

Results: Mechanical testing showed significantly greater stiffness in group I when compared to control (p < 0.05), and a dose dependent trend of increasing strength.

Radiographic analysis showed greater healing of fracture and callus formation in Group I compared to Groups II, III, and IV, at both three and five weeks post-fracture (P< 0.05).

Histological analysis showed an increase in bone formation in group I compared to the other groups.

Conclusion: This data from this model, suggests that TP508 enhances healing in high energy fractures. The results also suggest that the effects of TP508 are dose dependant, and are greater when delivered into the fracture site.

Once damaged, articular cartilage has limited capacity for self-repair due to their avascular and acellular nature. Tissue engineering approaches using cultured chondrocytes and biomaterials as scaffoldings hold promises for repairing cartilage defects. However, the source of articular chondrocytes is limited and the chon-drocytes may de-differentiate when cultured for a prolonged period. Bone marrow derived mesenchymal stem cells (BMSCs) have multi-differentiation potentials and autologous BMSCs are easy to obtain and culture with no/little immunological reaction when re-implanted.

24 NZW rabbits were used. Rabbit autologous BMSCs were obtained through marrow aspirations and expanded in culture under the chondrogenic induction media (DMEM, 10% FCS, plus 10ng/ml TGF-β1) for 3 weeks. A full-thickness articular cartilage defect (3 mm in diameter and 3 mm in depth) was created on both medial condyles in the rabbit. For experimental group (16 joints), the defects were filled immediately with alginate capsules containing autologous chondrogenic cells (8.5 x 104); for the control groups, the defects were filled with either alginate capsules alone (16 joints) or left untreated (16 joints). All the animals were terminated at 6 and 12 weeks after surgery and the cartilage samples were harvested for histology, immunochemistry and in situ hybridization examinations.

For histology, in the experimental group the defects were filled with immature hyaline-like cartilaginous tissues at 6 weeks; by 12 weeks the newly formed cartilage showing signs of remodeling and integrating into the surrounding articular cartilage. The expression of type II collagen in the newly formed cartilaginous tissues was confirmed by immunohistochemistry and by in situ hybridization methods. In the control groups, the defects were mainly filled with fibrous tissues in all the animals at the two time points examined. We have used Wakitani cartilage grading system for semi-quantitative histological evaluation. Significant lower scores (with superior histology) were found in the experimental group comparing to the two control groups.

Our results confirmed that full-thickness articular cartilage defects can be repaired by chondrogenically differentiated autologous BMSCs seeded into alginate capsules. Further studies are ongoing to explore the long term outcome of this treatment approach as well as using new scaffolds for cartilage tissue engineering.

The thrombin-related peptide, TP508, a synthetic 23 amino acid peptide, has been shown to promote soft tissue, cartilage and fracture repair. We have previously demonstrated that two injections of TP508 have signifi-cantly enhanced bone consolidation in a rabbit model of distraction osteogenesis. This study was to test if a single injection of TP508 in a slow-releasing preparation will have the similar effects.

Unilateral tibial osteoectomies were stabilized with M100 Orthofix lengtheners in 17 male adult NZW rabbits. After 7 days, lengthening was initiated at a rate of 1.4 mm/day for 6 days. The following treatments were given: Group 1: TP508 in saline (300ug/300ul, n=6) was injected into the osteotomy gap at day of surgery and into the lengthening gap at end of lengthening. Group 2 (Control): Dextran gel (300ul, n=6) and Group 3: 300ul Dextran gel mixed with microspheres containing 300ug TP508 (n=5), was injected into the lengthening gap at end of lengthening. All animals were terminated 2 weeks after lengthening. Bone formation was assessed by weekly radiography and the specimens were subject to pQCT, microCT and histology examinations.

On radiographies there was more bone formation in the TP508 treated groups than that of the control group at 1st week post-lengthening and complete union was seen in 50% rabbits in Group 1, 33% in Group 2, and 60% in Group 3 at termination. The mean BMD of the regenerates was significantly higher in the TP508 treated groups than that of the control group (p< 0.05). MicroCT analysis demonstrated advanced bone formation in the TP508 treated animals. For histology, the regenerates were mainly consisted of woven bone of neocortilization and callus remodelling in Groups 1 and 3, whereas in Group 2, focal defects with cartilaginous tissues were frequently seen.

In conclusion we have demonstrated that a single injection of TP508 in the form of slow releasing micro-spheres has enhanced bone consolidation during distraction osteogenesis. TP508 may therefore be applied in the slow-releasing preparation for augmenting bone formation at reduced doses, costs and risks of infections through repeated injections.

Mesenchymal stem cells (MSCs) are immunosuppressive and have been used to facilitate tissue repair in the context of allogeneic implantation. However, xenogeneic cell transplantation has not been fully explored. The present study investigated the feasibility of xenogeneic MSCs implantation in mice.

MSCs were harvested from the bone marrow of GFP rats (Green Fluorescent Protein transgenic rats), and cultured as previously described. 1 million GFP MSCs were loaded onto the synthetic HA/TCP porous Skelite blocks and implanted intramuscularly into the quadriceps of the MF1 and SCID mice. After 11 weeks, the implants were harvested and processed for histology examination. Upon termination, the mononuclear cells from the peripheral blood of each animal were also collected for mixed lymphocyte culture to examine lymphocyte proliferation potential and T-cell mediated cell lysis (cytotoxic) assays.

In the SCID mice, there was sparse osteoid tissue formation in the implants, whereas only dense connective tissues were seen in the implants of the MF1 mice. Osteocalcin mRNA expression was confirmed in the osteoid tissues in the implants from the SCID mice, but it was not detected in the MF1 mice by RT in situ PCR examination. Cells of GFP-rat origin were observed in both the MF1 and SCID mice (more so in the SCID mice) after 11 weeks implantation, which were confirmed by positive immunostaining of anti-GFP antibody. In the MF1 mice after 11 weeks xenogeneic MSCs implantation, the rate of lymphocyte proliferation was significantly increased when mixed with the GFP-MSCs compared to that of mixed lymphocyte culture assays in the SCID or MF1 mice without xenogeneic MSCs implantation, suggesting that implantation of xenoge-neic MSCs has promoted host anti-graft immunogenic responses towards to otherwise immunosuppressive MSCs.

In conclusion, xenogeneic rat MSCs transplanted in immunocompetent mice has survived for prolonged period, but their function was comprised to certain extent and this may be due to the increased host anti-graft immune sensitization after exposed to the xenogeneic MSCs.

Introduction: The aim of this research project was to establish a simple, reliable and repeatable externally fixed femoral fracture model. The rat was selected, as it was a suitable animal for use in a model of fracture repair and ovariectomy induced osteoporosis, both of which were to be investigated in future experiments. There are femoral fracture models described in the literature based on the insertion of an intramedullary nail prior to inducing a fracture. We felt, based on our experience of the unilateral externally fixed mouse fracture model, that external fixation would allow us to carry out radiographical and histological analysis of fracture healing without any of the tissue trauma caused by the insertion and removal of the intramedullary device.

Materials and Methods: A unilateral external fixator was chosen due to its simplicity. Four threaded stainless steel pins pass through holes in an aluminium plate with nuts placed on the pin above and below the plate. The holes in the plate were 0.1mm bigger than the pins and unthreaded allowing the plate to slide freely over the pins. Tightening of the upper nut compressed the plate against the lower nut holding the pin securely. 41 female Sprague-Dawley rats, aged between 12 and 18 weeks, were used. They were anaesthetised using a standard mixture of hypnorm and midazolam and analgesia, fluids and antibiotic were administered subcutaneously prior to surgery. The femur was exposed through a lateral approach and a standardised osteotomy was made prior to the application of the fixator plate. Accurate reduction was confirmed visually at the time of surgery and also by way of a post-op x-ray. 25 animals were sacrificed at 4 days and 1, 2, 4, 6 and 8 weeks for histology. The fractured limbs were harvested, fixed, decalcified and paraffin embedded as per standard protocol and serial sections were cut. These were stained with H& E and alcian blue and analysed 15 animals were sacrificed at 4,6 or 8 weeks for biomechanical strength testing. Four-point bending was carried out on freshly harvested femurs stored in normal saline between harvest and testing. Both limbs were tested and the fractured limbs were standardised relative to the unfractured limb. Maximum load to failure was recorded and stiffness was calculated from the load-displacement curve.

Results: No post-operative complications of fixation failure or infection occured. On histological assessment at D4 a predominantly lymphocytic inflammatory response was seen within the fracture haematoma. This inflammatory response was replaced with endosteal and periosteal new bone between wks 1 and 2. Bridging of the fracture gap was seen at week 6. Both stiffness and load to failure increased with increasing time. There was a statistically significant improvement in the percentage stiffness and percentage load to failure between 4 and 8 weeks (p=0.03 and p=0.018 respectively). The difference in load to failure between 6 and 8 weeks was also significantly different (p=0.042).

Discussion: A simple, reliable and repeatable externally fixed rat femoral fracture model has been established.

Introduction: The existence of circulating skeletal stem cells in the peripheral blood from different species including adult mouse and human has been found and documented. The circulating skeletal stem cells may provide a new source of stem cells that may be used for bone regeneration and tissue engineering applications. The aim of this study was to investigate the existence of circulating osteogenic stem cells in the rat peripheral blood, and to compare their osteogenic potentials with bone marrow mesenchymal stem cells (BMMSCs).

Methods: Whole blood from twelve female 3-month old SD rats was harvested by cardiac puncture and bone marrows were also collected. Mononuclear cells from both bone marrow and peripheral blood (PBMNCs) were isolated by Lymphoprep density gradient centrifugation method, and plated at a density of 300000 to 400000/cm2 in flasks with á-MEM medium and 15% FCS. The colony forming efficiency (CFE) was calculated after 10–14 days culture. The osteogenic, adipogenic, and chondrogenic differentiation potential of both BMMSCs and peripheral blood mononuclear cell subset were examined and compared under different specific culture conditions. In addition, both BMMSCs and peripheral blood mononuclear cell subset were seeded into absorbable porous calcium phosphate substitute and implanted subcutaneously into SCID mice for 12 weeks, and the implants were examined histologically.

Results: After 10–14 days in culture, the adherent fibroblast-like colonies were formed in the PBMNCs, with CFE ranging from 1.3 to 3.5 per 10000000 cells. Under osteogenic conditions, both BMMSCs and PBMNCs subset were positive for bone markers such as ALP, type I collagen and osteocalcin; bone nodules were formed in BMMSCs and PBMNCs subset long-term culture with positive Von Kossa and Alizarin Red S staining. Under adipogenic conditions, PBMNCs subset and BMMSCs were positive for Oil Red O and C/EBP á immunostaining. For chondrogenic differentiation studies, PBMNCs subset and BMMSCs were positive for type II collagen and they had Alcian blue positive nodules formation. After implantation with calcium phosphate substitutes in SCID mice for 12 weeks, osteoid and bony tissues were evident in the implants both loaded with PBMNCs subset and BMSCSs.

Conclusions: A subset of mononuclear cells that have multi-differentiation potential similar to BMMSCs exists in the rat peripheral blood. Our present study has shown that these circulating stem cells possess osteogenic potential in vitro and in vivo. Further work is ongoing to investigate the roles of PBMNCs subset in fracture healing and their recruiting and homing mechanisms.

NSAIDs inhibit fracture repair, yet the mechanism behind this effect is unknown. It is recognised that NSAIDs impede tumour growth via an inhibition of angiogenesis, primarily via a COX-2 pathway. We propose that the inhibition of fracture repair is via a similar mechanism and have investigated this hypothesis using a murine fracture model. 225 animals were randomised into either treatment (rofecoxib) or control groups and underwent a standard open femoral fracture treated using an external fixator. Outcomes measures involved assessment of healing using radiographic, histolological and biomechanical means; and measurement of blood flow across the fracture gap using Laser Doppler Flowmetry. X-ray analysis showed a similar healing pattern in both groups, however at days 16 and 32 the NSAID group had significantly poorer healing. Histological analysis showed that controls healed quicker (significant at days 24 and 32); and had more bone but less cartilage at day 8. Biomechanical testing showed controls were statistically stronger and stiffer at day 32, while NSAID animals had a significantly greater rate of fixation failure, leading to loss of pin-bone osseointegration; this occurred primarily before day 16. There was no difference in blood flow between the groups on the day of surgery, and both groups exhibited a similar flow pattern; NSAID animals however, exhibited a lower median flow from day 4 onwards, which was significantly poorer at days 4, 16 and 24. Positive correlations were demonstrated between a higher blood flow and both the histological and radiographic results. While NSAIDs were seen to inhibit fracture repair in all outcome measures; and were also noted to decrease blood flow at the fracture, with strong negative correlations being noted between NSAID prescription and fracture repair; multiple regression analysis suggest that this negative effect of NSAIDs on healing is independent of its inhibitory action on blood flow. COX-2 inhibitors are marketed as having cleaner side effect profiles and prescribing is on the rise. Recently however some of the newer COX-2 specific inhibitors have been removed from the market as their seemingly clean side effect profile has come under scrutiny. We have demonstrated that the COX-2 specific inhibitor rofecoxib does has a significant negative effect on fracture repair; and as hypothesised that it also has a significant negative effect on blood flow at the fracture site. While these outcomes strongly correlate, the mechanism behind the effect remains to be elucidated, as we have also demonstrated that these modalities are independent of each other.

Introduction: The existence of peripheral blood (PB) derived mesenchynal stem cells (PBMSCs) have been documented in several species including human. The circulating skeletal stem cells may provide a new source of stem cells that may be used for skeletal and other tissue engineering applications. The objective of this study is to further investigate and compare the biological characteristics of the PBMSCs with bone marrow derived MSCs in the GFP rats.

Methods: The peripheral blood (PB) from the GFP rats was harvested by cardiac puncture using syringes containing sodium heparin. Mononuclear cells were isolated by density gradient centrifugation method and plated at a density of 1–3~105/cm2 in flasks with D-MEM medium containing 15% FCS. The bone marrow (BM) was also collected for obtaining BMMSCs, the bone chips for osteoblastic cells, and the skin for skin fibroblasts. The phenotypes of the cells were characterized by immunocytochemistry (ICC), and flow cytometry methods. Gene expression profiles of 3-paired PBMSCs and BMMSCs cDNA samples were examined by Affymetrix gene chips microarray analysis. The multipotent differentiation potentials of PBMSCs into osteoblasts, chondrocytes, and adipocytes were examined under specific inductive conditions and checked with lineage specific markers. Finally, the osteogenic potential of the PBMSCs was examined by an in vivo implantation model in which the PBMSCs were seeded with HA-TCP powder complexes, and implanted subcutaneously in the severe compromised immunodeficiency (SCID) mice for 12 weeks, whereas the bone-derived osteoblasts and skin fibroblasts were used as controls.

Results: Compared with the BMMSCs, the PBMSCs shared some but not all common surface markers as demonstrated by (ICC) and flow cytometry examinations. The osteogenic differentiation of PBMSCs was defined with positive staining of type I collagen and osteocalcin; positive staining for alkaline phosphatase and Von Kossa staining for mineralized bone nodules. Adipogenic differentiation was evidenced by positive Oil red-O staining for accumulated lipids, and chondrogenic differentiation by positive type II collagen and Saferinin O positive staining. For gene expression profiles, in the Affymetrix chip general analysis, 83 genes were up regulated and 84 genes down regulated in the PBMSCs (vs BMMSCs, > 2 fold, E-B/B-E> 100, p< 0.05). Most of which genes are related to cell proliferation, differentiation, cytoskeleton, and calcium/iron homeostasis. After 12 weeks implantation in SCID mice, newly formed lamellar bone was clearly evident in the groups with PBMSCs implants, so as in the groups with osteoblasts implants, but only fibrous tissue was found in the group implanted with skin fibroblasts.

Discussion: This study demonstrated that the multi-potent PBMSCs in the GFP rats resemble BMMSCs in many aspects, but they are distinguishable from the BMMSCs in some biological characteristics and gene profiles. Our study has confirmed that these PBMSCs possess osteogenic potential in vitro and in vivo, suggesting that these circulating stem cells could serve as an alternative source as bone marrow derived MSCs for tissue engineering purposes.

During bone development and repair, angiogenesis, osteogenesis and bone remodeling (resorption) are closely associated processes with some common mediators involved. BMPs, VEGF and other cytokines are released from bone during bone resorption. Recent study showed that VEGF caused a dose- and time-dependent increase in bone resorption in vitro and in vivo, and BMP-2 markedly enhanced osteoclast differentiation induced by sRANKL and M-CSF in mouse osteoclast culture system. The aim of this study was to further examine the effects of VEGF and BMP-2 on osteoclastogenesis using in vitro human osteoclast culture system. Mononuclear cells were isolated by Lympo-Prep density gradient centrifugation from bone marrow washouts in bone samples from patients undergone total hip replacement. Mononuclear cells were plated at a density of 1 x 106/cm2 in a T-75 flask with aMEM and 15% FCS. The first medium change was made at day 7, when the floating cells were collected from the withdrawn media by centrifugation, and plated in a separate flask. The non-adherent cells in the 2nd flask were harvested again 24 hours later in a similar fashion. The non-adherent cells were then cultured in 24-well plates or calcium phosphate (Ca-P) coated plates, with osteoclast-inducing media (OC media) containing sRANKL 30 ng/ml and M-CSF 30 ng/ml, media were changed every 4 days. After 4 days culture in OC media, rhBMP-2 (3, 30, 300 ng/ml) and VEGF (25 ng/ml) were added respectively or in combination to the cell culture, and the culture was kept for total 16 days. The number of TRAP positive multinuclear cells in each well and the resorptive pit areas on the Ca-P coated plates were calculated and compared. Osteoclastic cell phenotype was defined by expressing tartrate resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and resorptive pit assay. By day 12–14, osteoclastic cells were found in all the experimental groups, they were positive for TRAP and VNR. The number of TRAP+ multinuclear cells were significantly reduced (p< 0.05, t-test) when rhBMP-2 (30 and 300 ng/ml) were present, and this was further reduced (p< 0.01) when rhVEGF was added together with rhBMP-2, comparing to the culture with OC media alone. Extensive lacunar resorption pits in the Ca-P coated plates were found in the culture treated with OC media and OC media with rhVEGF (25 ng/ml). The resorption pit areas were, however, significantly reduced when rhBMP-2 was added at 30 and 300 ng/ml with or without rhVEGF (25 ng/ml, p< 0.05, t-test). The presence of low concentration of rhBMP-2 (3 ng/ml) with VEGF had no effect on osteoclast number or the areas of resorption pit formation. In contrary to previous findings in the mouse osteoclast culture system, the present study had shown that the presence of rhBMP-2 at 30 and 300 ng/ml had strongly inhibited osteoclast differentiation and bone resorptive capability in the human osteoclast culture system, and the inhibition was further enhanced by the presence of rhVEGF. This study implies that VEGF and BMP-2 may be important, yet to be defined regulators, for osteoclastogenesis.

Introduction: Peripheral blood derived mesenchymal stem cells (PBMSCs) are multipotent cells capable of forming bone, cartilage, fat, and other connective tissues. Bone marrow derived mesenchymal stem cells (BMMSCs) have promoted repair a critical-sized bone defect in several animal models including mouse, rat, rabbit, and dog. The aim of this study was to investigate whether or not the use of allogenic BMMSCs and PBMSCs could regenerate a critical-sized bone defect in rabbit ulnae.

Methods: Rabbit peripheral blood mononuclear cells (PBMNCs) were isolated by density gradient centrifugation method and cultured at a density of 100,000/ cm2 in flasks with DMEM 15% FCS. Colony forming efficiency (CFE) was calculated and their multipotential differentiations into bone, cartilage, and fat were examined under different induction conditions. Specific differentiation markers were examined using cytochemistry and immunocytochemistry methods in the PBMSCs. Critical-sized ulna bone defects, 20 mm in length, were created in the mid-diaphysis of both ulnae in twelve 6 month old NZW rabbits. The ulnar defects were treated as the following 5 groups: empty control (n=4), PBMSCs/Skelite (multi-phase porous calcium phosphate resorbable substitute, EBI Company, USA) (n=5), BMMSCs/Skelite (n=4), PBMNCs/Skelite (n=5), and Skelite alone (n=5). All animals were sacrificed 12 weeks after treatment. The bone regeneration was evaluated by regular radiography, and all samples were subject to peripheral quantitative computed tomography (pQCT) and histological examination at the end point.

Results: The CFE of PBMSCs ranged from 1.2 to 13 per million mononuclear cells. Spindle and polygonal shaped cells were found in the primary PBMSCs colony, showing similar differentiation potential with BMMSCs. Mineralized bone nodules formed under osteogenic media were positive for Alizarin Red S staining in the PBMSCs. Chondrogenic differentiation was identified in serum free media containing TGF-¦Â1 (10 ng/ml), with type II collagen expression and Alcian blue positive nodule formation. Adipocytic differentiation was tested with or without adipogenic media, with positive Oil Red O staining for lipid accumulation and CEBP¦Á expression in the PBMSCs. After twelve weeks implantation, the ulnar defects were not healed in the empty control group; the total bone density in PBMSCs/Skelite and BMMSCs/Skelite treated defects were greater than that of PBMNCs/Skelite and Skelite alone treated groups (p< 0.05), with higher score of X-ray evaluation (p< 0.05). Histologically, there were a greater amount of new bone present in both the PBMSCs/Skelite and BMMSCs/Skelite treated groups compared to the PBMNCs/Skelite and Skelite alone treated groups.

Conclusions: This study demonstrated that PBMSCs were multipotent cells; allogenic PBMSCs loaded onto porous calcium phosphate resorbable substitute had enhanced bone regeneration of a critical-sized segmental defect in the rabbit ulna. PBMSCs may be a new source of osteogenic stem cells for bone regeneration and tissue engineering, and further investigations are undergoing to clarify their functions.

Introduction: Angiogenesis is essential during bone formation. Many studies have looked at the developing vascular network during normal and abnormal bone growth, using histological, immunohistological and contrast-radiological techniques; however all require sacrifice of animals to obtain tissue samples for examination and consequently chronological investigation of angiogenesis is not possible. We have endeavoured to produce an animal model, whereby quantitative assessment of blood flow, and callus formation across a fracture gap, can be repeatedly assessed.

Methods: The model is an adaptation of a 4-pin externally fixated murine femoral fracture previously developed in this department. Three extra conduits have been drilled onto the fixator cross-bar, such that it now links with an x-ray jig and implantable optical cable. The x-ray jig permits repeated lateral x-rays whereas the optical cable which is implanted adjacent to the fracture gap and connected to a laser, measures blood flow using the principle of the Doppler shift of light. Ten mice underwent surgery. Doppler readings and x-rays were taken on the day of surgery and subsequently at days 1, 2, 4, 8, 12, 16, 24 and 32.

Results: Fracture gap pixel density was seen to rise steadily and plateau at day 24, with significant statistical differences between the day of surgery and early time points, and then again between these early time-points (days 2, 4 and 8) and the late time-point day 24. Blood flow was noted to fall following the day of surgery and then slowly increase, with a rapid rise in flow at day 8 until day 16, when levels began to fall again to resting levels.

Conclusion: The data correlates with previous histo-morphological work performed in this department and also with early results from immunohistochemical studies. The above graph for blood flow conforms to that expected in a murine model of fracture healing, with a short initial drop in flow followed by a large rise as angiogenesis follows chondrocyte hypertrophy at the end of the first week, leading to callus formation. This in vivo model may be used to assess the effects on angiogenesis and callus formation of osteogenic compounds and investigate possible antiangiogenic mechanisms of action of medications such as NSAIDs that are known to be detrimental to fracture repair.

Introduction: Sufficient quantity of osteogenic cells is an essential aspect for a successful cell therapy in the treatment of difficult bone fractures and defects. At present, this was achieved by culturing bone marrow and bone-derived cells in a relatively long duration. A large number of the non-adherent mesenchymal stem cells were discarded during medium change. We hypothesize that collecting the non-adherent cells and re-plating them may result in more osteogenic cells in the same duration of cell culture. The aim of this study was to investigate the possibility of enhancing number of osteogenic cells by collecting non-adherent cells in the pull-off media and to examine their osteognic potentials.

Methods: Mononuclear cells were isolated by density gradient centrifugation method from bone marrow washouts in the bone samples obtained from 5 patients undergone total hip replacement. Mononuclear cells were plated at a density of 1 x 10⁶/cm² in a T-75 flask with αMEM medium and 15% FCS. The first medium change was made at day 7 and every 3 days thereafter. For the first three times of medium change, the removed media were centrifuged at 250 g for 10 minutes and plated in a separate T-75 (first time change) and T-25 flask (for the 2nd and 3rd times change). The non-adherent cells from the second and the third puff-off flasks were also collected and plated in separate T-25 flasks. Thus, 1xT-75 flask and 4xT-25 flasks of non-adherent cells resulted from the original T-75 flask. The cells in all flasks were harvested at 21 days from the day when the original flask was set up. The total number of cells in all pull off flasks were counted and compared with that of the original T-75 flask. Rate of cell proliferation with or without osteogenic growth medium were also examined by MTT method for passage 1 of both cells types. Osteogenic differentiation was defined with immunocytochemistry of bone markers: ALP, type I collagen, Osteocalcin and cbfa1. It is planed that cells of passage 2 will be mixed with HA powders and to be implanted into the SCID mice to examine the in-vivo osteogenic potential of these cells.

Results: Mesenchymal stem cells (MSCs) derived from the non-adherent population of human bone marrow culture have demonstrated having similar cell proliferation and differentiation potential in vitro, when compared to the MSCs derived from the adherent cell population. These cells expressed bone markers such as: ALP, type I collagen, osteocalcin and cbfa1. When the non-adherent cells were collected and cultured accumulatively, the total number of MSCs was increased to an average of 39.7% (36.6%–42.9%), compared to the number of cells obtained from the original T-75 flask.

Conclusions: Collecting the non-adherent cell population in the bone marrow culture appeared to result in more MSCs. This harvesting method may be used as a non-invasive way for enhancing MSC numbers in a given period of time. Further in vitro and in vivo studies of these MSCs of non-adherent origins may provide information for optimizing cell culture protocols for rapid expanding the osteogenic cells in vitro. This will facilitate the clinical applications of human osteogenic cell therapy.

Introduction: Recently, co-transplantion of mesenchy-mal stem cells (MSCs) with hematopoietic stem cells (HSCs) has been shown to alleviate complications such as GVHD and speeding recovery of HSCs. This in vivo finding suggests that coculture of MSCs and HSCs may enhance their growth potentials in vitro. As the large-scale expansion of HSCs has been achieved by NASA’s suspension culture system, we further examined the effects of this suspension culture system (rotary bio-reactor) on MSCs’ proliferation and differentiation potentials in vitro.

Methods: Mononuclear cell fractions (MNCs) of human bone marrow aspirates (n=6, ages 46–81) were collected by density gradient centrifugation. The cells were inoculated into bioreactor (RCCS, Synthecon Inc., Texas, USA) at the concentration 1x10⁶ cells/ml, in Myelocult^TM medium supplemented with 50ng/ml SCF, 20ng/ ml rhIL-3 and rhIL-6 (10ng/ml SCF, 2ng/ml IL-3 and IL-6 after the first feeding) and 10-6 M hydrocortisone for 8 days. The medium was fully exchanged after 3 days and 20% daily thereafter. Total cell numbers in the bioreactor were counted daily using hemacytometer. Cells from day 1, 4, and 8 cultures were subjected to tri-color flow cytometry examination using CD34, CD44, and Stro-1 antibodies. By the end of 8 day culture, the output cells were resuspended in DMEM medium with 10% FBS and cultured in T75 flasks at 1x10⁵ cells/cm2 for further 3 weeks. Upon harvest, half of the attached MSCs were prepared for western blotting assay using various antibodies. The other half was further cultured for 13–28 days in osteogenic, chondrogenic, and adipogenic induction medium respectively. Cell differentiation results were examined by histology staining, immunohistochemistry (ICC) and transmission electron microscope (TEM) examinations.

Results: After 8-day culture in bioreactor, flow-cytometric analysis confirmed that two cell populations, CD34+CD44+ (HSCs) and Stro-1+CD44+ (MSCs), increased 8-fold and 29-fold respectively, when compared to the values of the MNCs prior to bioreactor treatment. Cell counting revealed that the total cell expansion over 8 days was 9-fold above the number of the input MNCs. Western blotting data confirmed that bioreactor-expanded MSCs population remained in their early-stage with the expression of primitive MSCs markers such as CD105 (endoglin, SH-2) and Vimentin, whereas no expression of differentiation markers including osteocalcin (osteogenesis), Type II collegen (chondrogenesis) and C/EBPα (adipogenesis). Upon differentiation induction, the bioreactor-expanded MSCs were capable of differentiating into osteocytes, chondrocytes, and adipocytes as evidenced by histology staining, ICC and TEM examinations.

Discussion: Our study has shown that the percentage of MSCs (Stro-1+CD44+) increased 29 folds in the bone marrow derived MNCs after they have been cultured with Myelocult¢â medium in bioreactor for 8 days. The suspension culture system did not affect the subsequent in vitro proliferation and differentiation potentials of MSCs. Current study indicates that rotary bioreactor may be used to rapidly expand the numbers of traditionally attachment-dependent MSCs from bone marrow-derived MNCs, which may be very useful in clinical tissue engineering applications.

Dentin matrix protein (DMP-1), a phosphoprotein highly linked to dentin formation, has recently been reported to have an important role in skeletal development. Previously we reported that adult mice lacking the gene for DMP-1 exhibit the characteristics of chondrodysplasia, osteoarthritis, and showed severe defects in mineralization. DMP-1 knock-out (KO) mice display a profound defect in mineralization, and this is not due to a systemic defect in calcium/phosphate metabolism because serum levels of calcium and phosphate are similar to those in the wild-type mice. Although KO neonates and newborns appear normal, upon closer examination, these animals exhibit skeletal abnormalities, which include delayed secondary ossification and impaired bone remodelling. Heterozygous DMP-1 (H) mice however, show no apparent differences to the wild-type mice. In this study, biomechanical assessment tests of bones from DMP-1 KO mice were performed. Fifteen heterozygous, H, (DMP-1 +/−) and 15 KO, (DMP-1 −/−) male mice were produced and used in this study. At 1, 3 and 7.5 months of age, the mice were sacrificed and 4–5 ulnae from each animal group were harvested and stored in 70% ethanol solution. Volumetric density (BMD) measurements of the intact ulnae were performed using peripheral quantitative computed tomography (XCT960M; Stratec, Pforzheim, Germany) and Norland Stratec software version 5.10. One millimetre thick slices were scanned at a distance of 1 mm under the articular cartilage surface of the elbow as identified by the scout view of the CT scan. BMD of the corticalis and subcortical bone were recorded. Cross-sectional area measurements were also made at the mid-diaphysis of the ulnae. Biomechanical tests were performed in 3-point bending, with supports 3.5 mm apart at a rate of 3 mm/min (Lloyd Instruments Ltd, UK). The ultimate load, yield load and stiffness were determined from the load-displacement curves. All data were analysed using Mann-Whitney U tests (SPSS, Version 9, Chicago, Illinois). Differences were considered significant at p < 0.05. Density studies revealed that H mice had higher BMD than KO mice at all ages (p < 0.001). In the H and KO mice, the cortical BMD peaked at 3 and 7.5 months, respectively. At 1 month, the mean cross-sectional areas of the ulnae were larger in H mice compared to KO mice (0.50 mm2 Vs 0.33 mm2). However at 7.5 months of age, the reverse was observed (H = 0.75 mm2 and KO = 0.98 mm2). Biomechanically, stiffness increased with age at a higher rate in H mice than KO mice. Significant differences were observed at 3 months (p< 0.01) and 7.5 months (p< 0.05) between the two animal groups. There were no significant differences between stiffness values at 1 month. This study has demonstrated that DMP-1 deficiency leads to:

severely compromised bone mineralization;

Introduction: The existence of peripheral blood (PB) derived mesenchymal stem cells (PB-MSCs) have been documented in different mammalian species including young and adult human. However, the number of PB-MSCs is low in normal adult human blood. We have demonstrated previously that there was an increase in the number of PB-MSCs following long bone fracture and in the patients suffering from fracture non-union. The present study was to compare the biological characteristics of the PB-MSCs from fracture non-union patients, with human bone marrow derived MSCs (BM-MSCs).

Methods: 200 mls PB was collected from 9 patients suffering from fracture non-union. The mononuclear cells (MNCs) were isolated by density gradients centrifugation and cultured in á-MEM containing 15% FBS. The PB-MNCs from normal donors (n=8) and BM-MSCs from patients underwent total hip replacement were used as controls. The colony forming efficiency (CFE) of the PB-MSCs was calculated, and the phenotypes of PB-MSCs and BM-MSCs were compared using immunocytochemistry and flow cytometry methods. Their multipotent differentiation potentials into osteoblasts, chondrocytes, adipocytes, neurogenic and angiogenic cells were examined under specific inductive culture media. The in vivo osteogenic potential of PB-MSCs was examined by implanting the HA-TCP blocks seeded with PB-MSCs into the SCID mice for 12 weeks.

Results: After 28 days in culture, fibroblastic colonies were formed in the PB-MNCs cultures in 5 of 9 fracture non-union patients, with CFE ranging from 2.08–2.86 per 10^8 MNCs. No fibroblastic colony was seen in PB-MNCs cultures of the 8 normal donors. Under flow cytometry examination, PB-MSCs and BM-MSCs were CD34 (low) and CD105+, but PB-MSCs were CD29-, CD44-, and ALP (low), whereas BM-MSCs were CD29+, CD44+, and ALP (high). Under specific differentiation inductions, the PB-MSCs differentiated into osteoblastic cells (ALP+, type I collagen+, osteocalcin+ and Alizarin red+; chondrocytes (type II collagen+ and Alcian Blue nodules formation); adipocytes (Oil red-O positive lipid accumulation). Neurogenic differentiation was confirmed by positive neuro-filament staining, and differentiation into endothelial cells was evident with tube formation in 2D culture, and positive staining for VW factor and CD31. After implantation in the SCID mice for 12 weeks, newly formed woven bones were found in the biomaterials seeded with PB-MSCs, and they were positive for human osteocalcin immunostaining.

Discussion: This study indicated that there were more PB-MSCs in the peripheral circulation of the fracture non-union patients than that in the normal subjects. This may be due to a continous systemic response for recruiting MSCs from remote bone marrow sites, with attempt to repair the fracture(s). The PB-MSCs were clearly multi-potential cells, which had shared some common phenotypic markers with BM-MSCs, as well as many distinguishable makers from the BM-MSCs. The recruitment of the PB-MSCs through circulation might be a general phenomenon of systemic responses in many pathological conditions, such as fracture or wound healing and other systemic diseases. Further understanding the roles of PB-MSCs in diseases and repair may lead to novel therapeutic strategies.

Introduction: Fibroblast growth factor receptor 3 (FGFR3) is a tyrosine kinase membrane-spanning protein whose function is to regulate chondrocyte proliferation, differentiation and matrix production during cartilage development. Several mutations in FGFR3 have now been documented to link to human diseases. A number of these mutations result in constitutive activation of the FGFR3, leading to proliferation and premature differentiation of chondrocytes. Depending on the mutation and the resultant level of FGFR3 activation, mild to severe skeletal dysplasias such as achondroplasia (ACH), hypochondroplasia (HCH), thanatophoric dysplasia type I (TDI) and type II (TDII), and severe achondroplasia with developmental delay and acanthosis nigrans (SADDAN) may result. It has been postulated that the signalling pathways downstream of FGFR3 may be responsible for activating transcription factors, leading to up-regulation of cell cycle inhibitors and causing abnormal suppression of chondrocyte cell proliferation. However, the precise signalling pathways involved in FGFR3 mutation have as yet to be elucidated. The aim of this study was to investigate and compare the differences in the downstream signalling pathways between FGFR3 mutants.

Methods and Results: Wild type FGFR3 has been cloned into expression vector pcDNA3 and the construct has been used to generate four different FGFR3 mutants using site-directed mutagenesis. The mutations which have been introduced and the types of dysplasia they correspond to were as follows: K380R (ACH), N540K (HCH) and K650E (TDII). A kinase dead form of the receptor, K504R has also been generated. Wild type and each of the four mutant FGFR3 proteins in pcDNA3 vector have been successfully transfected into 293T cells using the calcium phosphate method. Immunoprecipitation and Western Blot analysis of cell lysates revealed expression of wild type protein in three isoforms of size 135kDa (mature), 120kDa (intermediate) and 98kDa (immature). The mutant proteins all followed a similar pattern of expression with the exception of the TDII mutant that did not express the mature form of the FGFR3. Changes in MAPK, PLCã and STAT 1 signalling pathways in response to FGFs-1, 2, 9 and 18 in the 293-cells of wild type and mutant forms of FGFR3 are now under investigation, in an attempt to define which pathways are mostly responsible for the resultant abnormal phenotypes.

Discussion: Genomics studies have demonstrated that FGFR3 expression is significantly upregulated during the osteoblastic differentiation of mesenchymal stem cells (MSCs) under BMP-2 stimulation in vitro. Subsequent functional studies have demonstrated that a selective ligand for FGFR3, FGF9, is able to induce tyrosine kinase signalling, and the osteoblastic differentiation of MSCs in vitro. Further understanding the signalling mechanisms of FGFR3 activation in normal and mutant forms may lead to discover potential anabolic agents that are based on FGFR3-FGFs pathways.

Introduction: Although distraction osteogenesis techniques have been used clinically for the treatment of many skeletal conditions with great success over the last 2 decades, one-step larger extent tibial lengthening (> 5 cm) still remains a clinical challenge. In which tension unbalance of bone and soft-tissue may occur, and complications such as foot drop, ankle and knee dysfunction, cartilage injure and secondary osteoarthritis were common. We have designed and manufactured a new lengthener, which allows bone and soft tissue to be lengthened in synchronism, and ankle joint remain in functional position and may move freely during lengthening.

Methods: A dynamic cross joint apparatus at ankle level was added to a classic Ilizarov circular four-ring lengthener, the apparatus is consisted of a half ring, two dynamic junctions and an elastic (spring) device. In application pins were inserted into distant and proximal segment of the tibia, also through calcanues, the external fixator with the trans-joint device was then applied. Total 296 patients (age 6–46, average 21), 466 legs, were treated with this new lengthener, among them were 55 cases of infantile paralysis, 38 cases of post-trauma bone defects, 33 cases with congenital dysplasia and 170 cases of chordrodysplasia, rickets, dwarf and short stature (height < 148cm). Unilateral tibia lengthening was performed in 126 legs and bilateral tibia lengthening was performed in 340 legs.

Results: Average lengthening for lower limb discrepancy cases was 6.8 cm (2–8cm), and 8.8 cm (8–18cm) for dwarf and short stature. Patients can stand straight and walk during the lengthening. Average movement of ankle joint remained at 10 degree in all cases and x-ray confirmed that average ankle joint space was 2.2 mm (1–4mm). There was no foot drop and ankle joint deformity seen, and in 98% cases ankle joint function fully recovered within 1.5 years after lengthening (6–8 months). Common complications were pinhole infection (25 cases) and broken pin (8 cases). If total lengthening was over 10cm, 70% cases developed slight ankle joint stiffness that would gradually recover after physiotherapy. Severe complications occurred in 5 cases (1%), including nonunion 1 case, mal-union 1 case, bone deformity 1 case and re-fracture 2 cases. All of those cases were cured with satisfactory clinical outcome.

Discussion: The challenge of larger range tibial lengthening is mainly the soft tissue complications, such as foot drop, varus and valgus deformity of ankle joint and loss of ankle function. Prolonged soft tissue traction around the ankle joint may lead to increasing cartilage compression, cartilage damage and partial or permanent loss of joint function. Our dynamic lengthener would allow synchronized lengthening of triceps, Achilles tendon and prosterior tibia muscle with tibia, maintain ankle joint space and free ankle movement. This device was simple and easy to apply, with no need of additional Achilles tendon lengthening. Our clinical study has demonstrated that this device drastically reduced the rate of soft tissue complication. This device makes larger extent tibial lengthening (> 5cm) safer and realistic in clinical practice.

The potential importance of bone morphogenic proteins (BMPs) to improve fracture healing is of great interest to orthopaedic surgeons. Although the complex mechanisms leading from the presence of local BMP (either endogenous or exogenous) to form bone is increasingly understood, however most appropriate time to administer exogenous BMP has yet to be elucidated. The purpose of this study was to investigate when BMP may be administered to a fracture arena in order to best improve fracture healing. Forty mice were randomised into 4 groups; (group I) control, treated at day 0 with placebo; (groups II, III and IV) treated with BMP at days 0, 4 and 8, respectively. All animals underwent a previously validated surgical procedure involving the creation of an open femoral fracture which is stabilised using a 4 pin external fixator. Thirty microlitres of bovine serum albumin (BSA) alone was used in group I, and the other groups (II, III and IV) were treated with a combination of the BSA and 2.5 microgrames of rhBMP-2. The BSA and rhBMP were injected through a lateral approach immediately after operation, or at 4, or 8 days postoperatively. At days 0, 8, 16 and 22, sequential radiographs were taken using a digital x-ray machine and at day 22 all animals were sacrificed. Both femora were harvested and assessed biomechanically in 3-point bending prior to fixation for histological evaluation. All data were analysed using Mann-Whitney U tests (SPSS, Version 9, Chicago, Illinois) and differences were considered significant at p < 0.05. X-ray analysis indicated that healing of fractures treated with BMP at day 0(group II) or day 4(group III) was significantly greater than that at both days 16 and 22 (p < 0.05) than those animals in placebo (group I) and BMP day 8(group V) treatment groups. Although the administration of BMP at day 4 seemed to cause more bone formation than treatment at day 0, no significant difference were observed. There were no differences between group IV and group I. Biomechanically, group III exhibited ultimate load values closest to the contralateral unoperated femora followed by group II, then IV and finally the control group I. Significant differences (p < 0.05) were observed between the control animals (group I) and both groups II and III. Qualitative histology suggested that at 22 days after surgery, only groups II and III had healed with woven bone. Group I and group IV had considerable amounts of fibrous tissue and cartilage at the fracture gap. This study suggests that a single percutaneous injection of BMP has a positive effect on fracture healing in this model, when prescribed between the time of injury (day 0) and 4 days. Data suggests that the most effective timing of delivery of BMP may not be at the time of surgery but actually in the early healing phase. The day 4 time point in the mouse model is likely to equate to that of 7–10 days in larger animals or humans. This suggests that current human treatment practices may require further investigation in order to elucidate the most appropriate time of delivery for these important proteins. This work may negate the current requirements for carrier products and large doses of these expensive drugs.

Introduction: Ten percent of fractures end in delayed or non-union. NSAIDs have been linked to an inhibitory action on fracture repair for three decades yet the mechanism of action remains to be elucidated. Cancer research has identified that NSAIDs impede cell proliferation by inhibiting angiogenesis. It is proposed that a similar mechanism occurs in the induction of NSAID induced non-unions. We have investigated this hypothesis in a randomized placebo control trial of the NSAID rofecoxib using a murine femoral fracture model.

Material and Methods: All animals had an open femoral fracture treated using an external fixator. Outcomes measures included x-ray, histology, and biomechanical testing, with laser Doppler used to assess blood flow across the fracture gap.

Results: Radiology showed similar healing patterns in both groups, however at the later stages (day 32) the NSAID group had significantly poorer healing. Histological analysis showed that controls healed quicker (days 24 and 32), with more callus (day 8) and less fibrous tissue (day 32). Biomechanical testing showed that controls were stronger at day 32. Both groups exhibited a similar pattern of blood flow; however NSAIDs exhibited a lower median flow from day 4 onwards (significant at days 4, 16 and 24).

Discussion: Positive correlations were demonstrated between both histological and radiographic assessments of healing, with increasing blood flow. NSAID animals exhibited lower flows, and poorer healing by all outcomes. Regression analysis demonstrates however that the negative effect of NSAIDs on fracture repair is independent of its inhibitory action on blood flow. In conclusion, COX-2 inhibitors are marketed as having cleaner side effect profiles and are widely used in trauma patients. Following development of a novel method of analyzing functional vascularity across a fracture gap, we have demonstrated that the COX-2 inhibitor rofecoxib has a significant negative effect on blood flow at the fracture gap as well as inhibiting fracture repair.

The thrombin-related peptide, TP508, is a synthetic 23 amino acid peptide, which represents the receptor binding domain of thrombin. TP508 mimics thrombin by interacting with receptors on cells involved in tissue repair. TP508 has been shown to enhance revascularization of injured tissue, and promote soft tissue wound healing, cartilage repair, and fracture repair. The aim of this study is to (1) test the effect of TP508 on bone regeneration during distraction osteogenesis; (2) study the chemotactic effect of TP508 on human osteoblasts.

Unilateral tibial osteoectomies were performed and stabilized with MX100 Orthofix lengthener in 5 male adult NZW rabbits. After 7 days, distraction was initiated at rates of 1.4 mm / day for 6 days. TP508 (100 μg/ml, n=2; 10 μg/ml, n=1) or saline (300 μl, n=2) was injected into the osteotomy / lengthening gap at days 1, 7 and 14 post surgery. Animals were sacrificed at 2 weeks after leg lengthening. Bone formation in the regenerate was assessed by radiography, quantitative computed tomography (pQCT) and histology. For chemotaxis studies, MG63 cells were cultured on glass cover slips for three days, and then inverted onto a Dunn chamber slide and sealed with dental wax. Gradients of TP508 (1, 10, 100 μg/ml) were added to the outer well and plain medium to the inner well. A sequence of images of the cells between the wells was taken via a CCD camera for 9 hours at interval of 10 minutes. Movements of individual cells were tracked and statistically analysed by a specially written Macro program. The Rayleigh test for unimodal clustering was used to determine the directional chemotactic movements.

The radiographic evaluation indicated a significant increase in new bone in the distraction regenerate in the TP508 treated groups at 1 and 2 weeks. pQCT images at 2 weeks demonstrated more advanced bone formation in the TP508 treated animals compared to the control. The mean total bone mineral density (BMD) of the regenerate, obtained from 3 slices was significantly greater (p = 0.019, t-test) in the TP508 treated group (BMD = 479.20 +/− 35.57 mg/ccm) than that in the saline control group (BMD = 355 +/− 2.83 mg/ccm). The histological evaluation supported the radiographic and the pQCT results. For chemotaxis study, no directional movements of the cells were found in the controls, whereas the MG63 cells were strongly chemotactic to TP508 at 1, 10 and 100 μg/ml concentrations.

This preliminary study shows that administration of TP508 enhances bone formation during distraction osteogenesis in the rabbit. The findings also show that TP508 has a chemotactic effect on osteoblasts, consistent with the effect of TP508 on fracture repair. A large animal study is in the process to confirm these findings and explore the underlying mechanisms.

Introduction: Osteoblasts precursors reside in the marrow and small numbers circulate in the blood. Our previous work demonstrated an increase in circulating cells following fracture in humans. Skeletal injury is recognised to stimulate a distant osteogenic response.

We hypothesised that in response to fracture, some integral osteoblasts are recruited via the circulation from remote bone marrow sites.

Method: We established a fracture union model in 3-month-old, male, New Zealand White rabbits and reimplanted labelled autologous osteoblast precursors. At date of submission we have 20 rabbits allocated into 4 groups. Three groups had labelled cells re-implanted, whilst the fourth control group did not receive cells. In groups I, II and III the cells were re-implanted into the fracture gap, into the circulation and into a remote bone marrow cavity respectively. There were six animals in groups I and IV, and four in both II and III.

All animals had bone marrow harvested from their right tibia by saline flush. The mononuclear cells were isolated and culture-expanded in osteogenic medium for 3 weeks. Fluorescent reporter molecules were incorporated into the cell membranes, 24 hours prior to re-implantation of the cells into the fracture model. A 3 mm ulnar defect was preformed in all the animals. In groups I–III this was established 48 hours prior to cell re-implantation.

The animals were sacrificed at least 3 weeks after fracture surgery. Representative samples of the fracture callous, lung, liver, spleen and kidney were harvested from all animals and cryo-sectioned. Using confocal microscopy, the labelled cells were expressed as the average in 5 high power fields for each solid tissue. In addition, cyto-spins were made from blood and marrow and the cell number expressed as a percentage of the total cells.

Results: In group I, labelled cells were identified in the fracture callous, establishing their viability in vivo. Following intravenous re-implantation a smaller number of labelled cells were identified in the callous. When the cells were re-implanted into a remote marrow site, the number of cells in the callous was greater than after venous reimplantation, but less numerous than those in group I.

In all sections, these labelled cells appeared on trabecular surfaces in an osteoblastic fashion, but occasionally they were surrounded by osteoid, corresponding to osteocytes.

A small number of labelled cells were found in the blood, bone marrow, lung, liver and spleen of all animals in groups I–III. No labelled cells were identified in the kidney tissue.

Discussion and Conclusions: We have demonstrated that cells from remote sites are integral in fracture healing. Their presence in callous following venous administration supports recruitment via the circulation. This preliminary data is a proof of concept. This is an exciting new phenomenon, which could provide alternatives for harvesting skeletal progenitor cells and for their delivery in the treatment of bony pathology.

Introduction: The potential of Bone Morphogenic Protein (BMP) to improve fracture healing is of great interest to orthopaedic surgeons. Although the complex mechanisms leading from the presence of local BMP to the fracture scenario has yet to be elucidated. The purpose of this study was to investigate whether introducing rhBMP-2 to the fracture arena, some days after fixation could be more beneficial to fracture repair.

Methods: 40 animals were randomised into 4 groups; namely control treatment at day 0 or rhBMP-2 treatment at days 0, 4 or 8 post-surgery. All animals underwent a previously validated surgical procedure involving creation of an open femoral fracture fixed using a 4-pin external fixator. 30 μl of bovine serum albumin (BSA) alone (control) or mixed with 2.5 μg of rhBMP-2 (treatments) was injected via a lateral approach directly into the fracture gap, either following closure of the wounds (day 0) or at 4 or 8 days postoperatively. Animals were assessed as to the outcome of surgery by digital sequential x-ray at days 0, 8, 16 and 22 using a Faxitron MX-20 camera; and y either biomechanical testing under a 3-point bending technique (Lloyd Instruments Ltd, UK) or histological examination following sacrifice at day 22. Data were analysed using Mann-Whitney U and Wilcoxon Tests for statistical differences (SPSS, Version 9). Differences were considered significant when p< 0.05.

Results: X-ray analysis indicated that healing of fractures treated with rhBMP-2 at day 0 or day 4 was significantly greater than the two other groups at days 16 and 22. BMP given at day 4 tended to a greater effect than when given at day 0, though the range was too great to show a statistical difference. There were no differences between the BMP-8 and the BSA control groups. Mechanical testing showed that only animals that had received rhBMP-2 at day 4 had attained similar peak loads to failure to those of their contralateral unoperated leg. Bones from animals receiving rhBMP-2 at day 0 had attained the next greatest strength, which was followed by rhBMP-2 administration at day 8 animals, whereas the animals receiving BSA attaining the least strength. There was a statistical difference (p< 0.05) between both rhBMP-2 day 4 and day 0 groups compared to the BSA control group. Qualitative histology suggested that the rhBMP-2 day 0 and day 4 groups had almost fully healed with new bone whereas the BSA and rhBMP-2 day 8 groups still had considerable mounts of fibrous tissue and cartilage at the fracture gap 22 days following surgery.

Conclusions: The study demonstrates that a single percutaneous injection of rhBMP-2 has a positive effect on fracture healing, when prescribed at the time of injury or during the early period of fracture repair. Data suggests that the most effective timing of delivery of BMP may not be at the time of surgery but in the early healing phase. The day 4 time point in the mouse model is likely to equate to that of 7–10 days in larger animals or humans. Further investigation as to the most appropriate time for intervention using these proteins is warranted and may negate the current requirement for carried products and large doses of these expensive drugs.

Introduction: Bone mineral density (BMD) is currently the gold standard in predicting osteoporotic fracture, but evidence suggests that over one third of such fractures occur in those with osteopenia or even normal BMD. The level of bone turnover may affect bone quality in these patients independently of BMD. Bone markers have evolved as tools in monitoring anti-resorptive treatment in osteoporosis.

Aims: The aim of this study was to investigate if levels of bone markers in postmenopausal women could be used as an adjunct to BMD measurements in the assessment of fragility fracture risk.

Patients and Methods: 60 postmenopausal women (30 osteoporotic, 30 with normal BDM) were studied. A single BMD measurement by dual energy x-ray absorptiometry (DEXA) enabled categorisation. Serum bone formation markers (bone specific alkaline phosphatase (BSAP) and osteocalcin (OC)), and resorption marker (C-telopetide of type 1 collagen (CTX)), were measured. History of low trauma fracture was documented for each woman.

Results: 36% of the osteoporotic group had experienced at least one fragility fracture. However, the femoral neck and combined spinal BMD in these women was not significantly different from the 64% of osteoporotic women who had no prior fracture. There was also no significant difference in the age of women in both subgroups. Serum bone markers were significantly increased in the osteoporotic fracture subgroup when compared to the non-fracture subgroup and also to the non-osteoporotic controls. The largest increases were seen in the levels of CTX. Smaller increases in all markers were seen when the non-fracture subgroup was compared to the non-osteoporotic control group but these increases did not reach statistical significance.

Conclusions: Bone turnover is significantly increased in postmenopausal osteoporotic women with previous fracture compared to both osteoporotic non-fracture counterparts and non-osteoporotic controls. This suggests higher bone turnover will increase fracture risk in osteoporotic women. It is possible that combining 2 or 3 markers to produce an “index of bone turnover” would be a useful tool when used in addition to BMD to identify those at greatest fracture risk.

Background and Hypothesis: High-energy fractures associated with severe soft tissue injury have a significant incidence of delayed or non-union. The soft tissue envelope may adversely contribute to the healing of a fracture, not only in stripping of the periosteal blood supply, development of compartment syndrome or tissue interposition between the bone ends but also in its ability to generate an intense acute inflammatory response. Inflammation is the initiator of healing; in clinical scenarios of impaired inflammation (immune deficiency, NSAIDs, corticosteroids) healing is delayed; interestingly, in injury with excess inflammation (CVA, MI) healing is also delayed. Would the inflammatory response following high-energy fractures contribute beneficially or adversely to the healing of the underlying fracture? Using an in-house murine femoral fracture model which reliably demonstrated features of delayed fracture healing when associated with a severe overlying muscle crush injury we proposed these hypotheses:

That fracture callus with overlying muscle crush would contain raised expression of acute inflammatory cytokines (IL-1β, IL-6 and TNF-α).

Methods: Total RNA was extracted from normal fracture callus (FO) and muscle crush fracture callus (MC) at day 2, day 4 and day 8. Semi-quantitative RT-PCR was used to compare IL-1β, IL-6 and TNF-α mRNA expression. Histomorpometric analysis of ICC stained sections of the FO and the MC groups was used to estimate IL-1β, IL-6 and TNF-α protein expression within the callus. Positively staining areas for the cytokine within the callus were a semi-quantified and compared between groups. Finally, blocking antibodies to IL-1β and TNF-α were injected into MC fracture callus at day 0, 4 and 8. Control MC group had vehicle only injected. Fracture healing was measured using radiological, histomorphological and biomechanical outcome measures. Following a pilot dosing experiment, the effect of blocking antibodies on fracture healing was compared between MC and MC with antibody groups.

Results: The MC group IL-1β mRNA expression was significantly higher than FO at day 4 and day 8 (p=0.05). ICC for IL-1β protein expression was higher on day 4 and on day 8 in the MC group, significant at day 8 (p=0.03). TNF-α mRNA expression in the MC group at day 8 was significantly higher than the FO group (p=0.05). ICC for TNF-α protein in the MC group peaked at day 8 and was significantly higher than the FO group (p< 0.03). IL-6 mRNA expression was significantly raised in the MC group at day 4 and 8 compared with the FO group (p=0.05). ICC for IL-6 protein showed significantly increased expression at day 8 in the MC group (p=0.05). The patterns of expression of the mRNA and proteins were similar.

Injection of anti-TNF-α antibodies into MC mice caused more new bone formation on day 16 (p=0.03) and day 24 (p=0.06), stiffer calluses at day 24 (p=0.01) and faster fracture gap obliteration at day 16 (p=0.05) and day 24 (p=0.001). IL-1β blockade had slightly less effect, more new bone formationd ay 16 (p=0.01) and day 24 (p=0.03), slightly stiffer (p=0.08), but no significant difference in fracture gap obliteration from controls.

Conclusion: The effect of muscle crush around the fracture callus was to increase and prolong the expression of inflammatory cytokines with the callus. The effect of blocking these excessive inflammatory cytokines in our model was to improve fracture healing. Excessive inflammatory cytokines (IL-1β, IL-6, TNF-α) in bone impair new bone production by osteoblasts, inhibit the recruitment and differentiation of mesenchymal precursors and promote osteoclastogenesis. The mechanism of action of blocking antibodies may be due to inhibition of the antiosteogenic effects of these cytokines.

Introduction: 5–10% of all fractures end in delayed or non-union. It has been reported for 3 decades that NSAIDs have an inhibitory action on fracture repair, yet GPs still prescribe these drugs in up to 50% of fracture patients. Not all fracture patients who are treated with this class of medication go on to develop non-union, yet a strong correlation has been shown in clinical studies between long bone fractures and development of delayed and non-union. The mechanism behind this effect has yet to be elucidated. In cancer research it has been shown that NSAIDs, primarily by a COX-2 pathway, can exert an inhibitory action on cell proliferation by inhibiting angiogenesis. It is proposed that a similar mechanism occurs in the induction of NSAID induced delayed fracture repair. We have investigated this hypothesis using an externally fixated murine model of femoral fracture.

Methods: 158 animals were randomised into either treatment (Rofecoxib 5mg/kg/day in a 0.5% methylcellulose carrier) or control (carrier alone). All had a standard surgical regimen involving creating of an open femoral fracture with treatment using a 4-pin external fixator under the isoflurane inhalational anaesthesia. Outcome measures included standardised x-rays (Faxitron MX-20) and Laser Doppler Flow (Oxford Optronics) measurements taken at days 0, 4, 8, 16, 24 and 32, along with biomechanical testing (Lloyd Instruments Ltd) at days 24 and 32. Data was entered into a spreadsheet and analysed using Mann-Whitney U and Wilcoxon Tests for statistical differences (SPSS, Version 9), with statistical significance being attained when p< 0.05.

Results: A greater number of animals in the NSAID group had a failure of treatment with loss fixation due to pin pullout from poor osseointegration (53% NSAID compared to 26% controls). Of those animals that completed the studies, x-ray analysis showed a change in pixel density at the fracture gap suggesting poorer healing of the NSAID animals that was statistically different at days 16, 24 and 32. Biomechanical testing suggested treatment animals had attained statistically less peak loads and stiffness at day 32. Laser Doppler Flow measurements across the fracture gap showed generally less flow at all time points in the NSAID group. This was statistically significant at days 4 and 24.

Conclusions: The new selective and specific COX-2 inhibitors are marketed as having a cleaner side effect profile and are being widely used by primary care practitioners in trauma patients. Not all animals that are treated with NSAIDs go on to develop a delayed union and some are able to heal with similar mechanical properties to animals in a control group. However, we have illustrated that the highly specific COX-2 inhibitor rofexcoxib has a significant negative effect on maintenance of fracture fixation and fracture repair in this model, both in terms of x-ray and biomechanical analysis. We have also shown that the inhibition of fracture healing is associated with a decrease in blood flow at the fracture site leading to the hypothesis that the mechanism behind the effect is via an inhibition of angiogenesis.

Introduction Posterior cruciate ligament (PCL) reconstruction has been shown to restore the posterior stability of the knee during posterior drawer tests. However, we have previously published a report showing that a PCL deficient knee has abnormal rotation under load. We hypothesise that a PCL reconstruction does not restore rotational stability to the knee.

Methods In-vitro kinematics under simulated muscle loads after PCL reconstruction were measured. Eight fresh-frozen cadaveric knees were tested on a robotic testing system. The system applied a posterior drawer of 130N and a combined quadriceps/hamstrings load (400N/200N) at 0°, 30°, 60°, 90°, and 120° of flexion. Tibial motion with respect to the femur was measured with the PCL intact, resected and reconstructed using an Achilles tendon allograft. Posterior tibial translation (PTT) and internal/external rotation were analyzed using a repeated measures ANOVA.

Results PCL deficiency significantly increased (p< 0.05) PTT under posterior drawer. Reconstruction significantly reduced the increased PTT to the level of the intact knee at all flexion angles. Under the muscle load, the deficiency resulted in significantly higher PTT at 60 to 120, and reconstruction did not significantly reduce the increased PTT. PCL deficiency significantly increased external rotation at 90° and 120°. PCL reconstruction did not significantly reduce the increased external rotation caused by PCL deficiency.

Conclusions Under simulated muscle loading, PCL reconstruction did not restore the translation and rotation of the tibia, despite restoring posterior stability under posterior drawer. Our data may help to identify the biomechanical factors that lead to the long-term development of osteoarthritis following PCL injury and reconstruction.

In relation to the conduct of this study, one or more of the authors is in receipt of a research grant from a non-commercial source.

During the process of distraction osteogenesis new bone is formed rapidly and undergoes remodelling almost immediately. Little is known about the regulatory mechanisms governing the removal of the redundant callus in this process. Tissue homeostasis is achieved by a delicate balance between the processes of cell death (apoptosis) and cell proliferation. The aim of this study was to test the hypothesis that apoptosis is involved during distraction osteogenesis.

Mid-tibial osteotomies were performed in 6 adult male NZW rabbits (age; 24 weeks, weight; 3.0 −3.5 kg), and the tibiae stabilised with unilateral external fixators (Orthofix M-100). 7 days later, twice daily distraction was initiated at rates of 0.7 mm/day for 3 weeks. BrdUrd (40mg/kg) was injected intravenously to the rabbit 1h before killing. The regenerate bone was collected, fixed in 10% buffered formalin and decalcified for paraffin embedding. Some fresh regenerate bone tissues were also prepared for examination under transmission electronic microscopy (TEM). BrdUrd immunohistochemistry has been used to detect proliferating cells and the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end-labelling (TUNEL) method was used to identify cells undergoing apoptosis. To detect bone-resorbing cells, tartrate-resistant acid phosphatase (TRAP) staining was also performed.

BrdUrd positive cells and TUNEL-positive cells were shown to coexist in most of the areas in the regenerates. In the mineralisation front, the majority of the TUNEL-positive cells were present in the transitional region between the fibrous tissue and the new bone. The TUNEL-positive cells were close to or on bone surfaces, and some of the newly formed osteocytes in the new trabeculae were also positive. The TUNEL-positive cells were also seen in the cartilage region of the regenerate. However, the TUNEL labelling was greatly reduced in the new bone close to the osteotomised bone ends, TUNEL-positive labelling were not detected in the cortical bone of the osteotomised bone ends and in the adjacent surrounding periosteum. TRAP staining in the regenerate revealed similar patterns of distribution to those of the TUNEL staining. There were more TRAP-positive cells in the new bone near the mineralisation front than in that of the new bone region, which was close to the osteotomised bone ends. TEM examinations have demonstrated characteristic signs of apoptotic changes in the fibroblast, osteoblast and osteocytes in the specific regions of the distraction regenerate.

The study provided evidence that in the process of rapid bone formation during distraction osteogenesis, superfluous cells are removed by apoptotic mechanisms. The demonstration of a mixture of proliferative and apoptotic cell populations in the regenerating tissue, indicates that apoptosis and cell proliferation may be regulated by local factors. The neovascularisation of the regenerate and withdrawal of growth factors and cytokines may be responsible for apoptosis occurring in some parts of the regenerating tissue. The changes of distribution of apoptotic cells in the different regions of the regenerate, together with the observed patterns of osteoclast activities, suggest that bone cells undergoing apoptosis may initiate rapid bone remodelling seen during distraction osteogenesis.

Fracture repair is a complex physiological process during which bone shows the remarkable ability to mount a repair process, restoring its mechanical integrity and anatomical configuration by original osseous tissue. Programmed cell death, or apoptosis, is a naturally occurring cell suicide pathway with a homeostatic function in the maintenance of continuously renewing tissues. The present study investigated the relation between cell proliferation and cell death (apoptosis) during fracture healing in a mouse femoral model.

Left femoral osteotomies were performed in 20 male CFLP mice (35–45g), immobilised with uniplanar external fixators. 4 animals were sacrificed on days 2, 4, 8, 16 and 24 post-fracture and fracture callus collected for paraffin embedding. Localisation of cell proliferation was examined using immunohistochemistry with proliferating cell nuclear antigen (PCNA) monoclonal antibody. Apoptotic cells were visualised with the terminal deoxynucleotidyl transferase (TdT)–mediated dUTP-biotin nick end-labelling (TUNEL) method. Random images of each time specific specimen were captured via a digital camera and the positive labelling indices of PCNA and TUNEL labelling were calculated and statically compared.

Cell proliferation and apoptosis were found co-existing during the entire period of fracture healing studied. Cell proliferation was predominant in the early phases of fracture healing (days 2–8). PCNA positive labelling index peaked at day 8 (p< 0.01, t-test) and PCNA-positive cells were not limited to the fracture gap mesenchymal tissues but extended in the periosteum along most of the fractured femur. TUNEL positive labelling was minimal in the early stages (days 2–8). In later stages of fracture healing (days 16–24), PCNA expression declined as intramembranous and endochondral ossification spread within the fracture site and apoptosis was the dominant cell activity with the TUNEL positive labelling index peaked at day 16 (p< 0.05, t-test) and then declined sharply at day 24.

The current study indicated that apoptosis was a normal concomitant during fracture repair, confirming programmed cell death in chondrocytes and bone cells, and that cell proliferation and apoptosis were tempero-spatially dependent. These findings support the view that apoptosis is a natural process, genetically programmed and active during fracture repair. The demonstration of a mixture of proliferative and apoptotic cell populations in the regenerating tissues of fracture callus, suggests that apoptosis and cell proliferation may be regulated by local factors during fracture healing.

NSAID’s cycle-oxygenase (COX) inhibitory characteristics are either non-specific, COX-1 preferential or recently COX-2 preferential. NSAID’s have been widely reported to delay fracture repair however the mechanism of this affect remains unclear.

Left femoral osteotomies were performed in 54 male 3 month old CFLP mice immobilised with uniplanar external fixators. 27 externally fixated mice received 4mg/kg meloxicam,b.d., from the day of surgery, by gavage. The control group received the carrier alone. 18 mice had external fixators applied to intact femurs and received no meloxicam as a sham control. Individual mouse movement, was quantified each day by autocounters using an infrared beam motion detection system. Plasma was obtained by right ventricular aspiration under anaesthesia on days 2,4,8 and 16-post surgery.

A validated bioassay and a slot blotting immunoassay were employed to determine the plasma concentration of 11-6 and relative TNF-α levels to normal mouse serum.

TNF-α levels peaked at day 4 and were suppressed by COX-2 inhibition. Both the control and treatment groups had higher levels of TNF-α than the non-fractured controls. The plasma concentration of 11-6 was elevated by COX-2 inhibition at all time points. The levels of TNF-α and 11-6 correlated in fracture control and treatment groups (Spearman’s 0.039 and 0.002 respectively). The 11-6 plasma concentration correlated to the animal motion in the treatment group alone (Spearman’s 0.017).

As it has been shown that TNF-α induces 11-6 production and that this inhibits TNF-α production a possible model for these interaction is shown below.