Background: The use of fresh morsellised allograft in impaction bone grafting for revision hip surgery remains the gold standard. Bone marrow contains osteogenic progenitor cells that arise from multipotent mesenchymal stem cells and we propose that in combination with allograft will produce a living composite with biological and mechanical potential. This study aimed to determine if human bone marrow stromal cells (HBMSC) seeded onto highly washed morsellised allograft could survive the impaction process, differentiate and proliferate along the osteogenic lineage and confer biomechanical advantage in comparison to impacted allograft alone

Methods: HBMSC were isolated and culture expanded in vitro under osteogenic conditions. Cells were seeded onto prepared morsellised allograft and impacted with a force equivalent to a standard femoral impaction (474J/m2). Samples were incubated for either two or four week periods under osteogenic conditions and analysed for cell viability, histology, immunohistochemistry, and biochemical analysis of cell number and osteogenic enzyme activity. Mechanical shear testing, using a Cam shear tester was performed, under three physiological compressive stresses (50N, 150N, 250N) from which the shear strength, internal friction angle and particle interlocking values were derived.

Results: Cell viability of HBMSC post impaction, was confirmed with cell tracker green staining, a marker of viable cells, and observed throughout all samples. There was a significant increase in DNA content and specific alkaline phosphatase activity compared to impacted seeded allograft samples. Immunohistochemical staining for type I collagen confirmed cell differentiation along the osteogenic lineage. Mechanical shear testing demonstrated a statistical significant increase in shear strength and interparticulate cohesion in the allograft/hBMSC group over allograft alone at 2 and 4 week intervals (p< 0.001).

Conclusion: HBMSC seeded onto allograft resulted in the formation of a living composite capable of withstanding the forces equivalent to a standard femoral impaction. HBMSC under osteogenic conditions were observed to differentiate and proliferate along the osteogenic lineage. In addition, an allograft/HBMSC living composite confers a biomechanical advantage over allograft alone These changes resulting in enhancement of biological and mechanical properties of bone graft within impaction bone grafting have implications for translation and future change in orthopaedic practice in an increasing ageing population.

The use of fresh morsellised allograft in impaction bone grafting for revision hip surgery remains the gold standard. Bone marrow contains osteogenic progenitor cells that arise from multipotent mesenchymal stem cells and we propose that in combination with allograft will produce a living composite with biological and mechanical potential. This study aimed to determine if human bone marrow stromal cells (HBMSC) seeded onto highly washed morsellised allograft could survive the impaction process, differentiate and proliferate along the osteogenic lineage and confer biomechanical advantage in comparison to impacted allograft alone. Future work into the development of a bioreactor is planned for the potential safe translation of such a technique into clinical practice.

Methods: HBMSC were isolated and culture expanded in vitro under osteogenic conditions. Cells were seeded onto prepared morsellised allograft and impacted with a force equivalent to a standard femoral impaction (474J/m2). Samples were incubated for either two or four week periods under osteogenic conditions and analysed for cell viability, histology, immunocytochemistry, and biochemical analysis of cell number and osteogenic enzyme activity. Mechanical shear testing, using a Cam shear tester was performed, under three physiological compressive stresses (50N, 150N, 250N) from which the shear strength, internal friction angle and particle interlocking values were derived.

Results: HBMSC survival post impaction, as evidenced by cell tracker green staining, was seen throughout the samples. There was a significant increase in DNA content (P< 0.05) and specific alkaline phosphatase activity (P< 0.05) compared to impacted seeded allograft samples. Immunocytochemistry staining for type I collagen confirmed cell differentiation along the osteogenic lineage. There was no statistical difference in the shear strength, internal friction angle and particulate cohesion between the two groups at 2 and 4 weeks.

Conclusion: HBMSC seeded onto allograft resulted in the formation of a living composite capable of withstanding the forces equivalent to a standard femoral impaction and, under osteogenic conditions, differentiate and proliferate along the osteogenic lineage. In addition, there was no reduction in aggregate shear strength and longer term studies are warranted to examine the biomechanical advantage of a living composite. The therapeutic implications of such composites auger well for orthopaedic applications.

Introduction: Bone is unique with a vast potential for regeneration from cells with stem cell characteristics. With an increasing aging population, clinical imperatives to augment and facilitate tissue repair have highlighted the therapeutic potential of harnessing mes-enchymal populations from bone. We describe laboratory and clinical findings from two clinical cases, where different proximal femoral conditions (AVN, bone cyst) were treated with impacted allograft augmented with marrow-derived allogeneic progenitor cells.

Methods: Marrow was aspirated from the posterior superior iliac crest and seeded onto prepared washed morsellised allograft. The seeded graft was left for 40 minutes to allow adherence of the marrow-derived osteoprogenitor cells prior to impaction into the defect. Samples of the impacted graft were taken for in-vitro analysis of cell viability, histology and biochemical analysis of cell number and osteogenic enzyme activity. The total force imparted during impaction was calculated using a load cell, with three independent surgeons performing a laboratory simulation of the impaction technique.

Results: Both patients made a rapid clinical recovery after an overnight stay. Imaging confirmed filling of the defects with increased density on plain radiographs suggesting good impaction of the graft composite. Immu-nohistochemical staining of graft samples demonstrated that a living composite graft with osteogenic activity had been introduced into the defects as evidenced by cell tracker green viability and alkaline phosphatase (osteogenic marker) expression and specific activity. The average peak forces during impaction were 0.7kN corresponding to average peak stresses within the graft of 8.3MPa. Similar forces were seen between operators.

Discussion: Replacement of bone loss remains a major challenge in orthopaedic practice. Although allograft remains the gold standard where large volumes preclude autograft, allograft has little osteoinductive potential. We demonstrate that marrow-derived cells can adhere to highly washed morsellised allograft, survive the impaction process, and are of the osteoblastic phenotype creating a living composite. Thus we conclude, impacted allograft seeded with autologous marrow cells allows the delivery of a biologically active scaffold for the treatment of bone deficiency. In addition this is a novel straightforward technique, surgeon independent and with applications in a number of orthopaedic scenarios.

The ability to generate replacement human tissues on demand is a major clinical need. Indeed the paucity of techniques in reconstructive surgery and trauma emphasize the urgent requirement for alternative strategies for the formation of new tissues and organs. The idea of biomimesis is to abstract good design principles and optimizations from nature and incorporate them in the construction of synthetic materials and structures. Direct appropriation of natural inorganic skeletons is also biomimetic since their unique properties inform us on ways to generate functional, optimized scaffolds.

A number of well characterized natural skeletons were investigated as potential scaffolds for tissue regeneration using mesenchymal stem cell populations. Marine sponges, sea urchin skeletons and nacre were found to possess unique functional properties that supported human cell attachment, growth and proliferation and provided organic/ inorganic extracellular matrix analogues for guided tissue regeneration.

A good understanding of the processses involved in biomineralisation and the emergence of complex inorganic forms has inspired synthetic strategies for the formation of biological analogues (organised inorganic materials with biological form). We have developed two functional examples of biological structures generated using biomimetic materials chemistry with applications for human tissue regeneration. Mineralised biopoly-saccharide microcapsules provided enclosed micro-environments with an appropriate physical structure and physiological milieu, for the support of the initial stages of tissue regeneration combined with a capacity to deliver human cells, plasmid DNA and controlled release of biological factors such as cytokines. Calcium carbonate porous microspheres analogous to microscopic coccolithophore shells provided a template for tissue formation and a mechanism for the delivery of DNA and functional biological factors. These biomi-metic structures have considerable potential as scaffolds for skeletal repair and regeneration, particularly when combined with inductive and stimulatory biological factors (cytokines, morphogens, signal molecules) and plasmid DNA carrying with them chemical cues that modulate and direct permanent tissue formation complimentary with the host.

Clonal chondrocytes of osteoarthritic (OA) cartilage express an aberrant set of genes. We hypothesize that this aberrant gene expression may be due to clonally inherited epigenetic changes, defined as altered gene expression without changes in genetic sequence. The major epigenetic changes are due to altered DNA methylations in crucial parts of the promoter region. If the cytosines of CpG dinucleotides are methylated, the gene will be silenced, even if the right transcription factors are present. Similarly, de-methylations may activate previously silenced genes. Our aims were to provide ‘proof-of-concept’ data by examining the methylation status of genes in OA vs non-OA chondrocytes. Articular cartilage was obtained a) from the cartilage of fracture-neck-of-femur (#NOF) patients and b) from or around the eroded regions of OA samples. The former was full thickness cartilage, the latter was partially degraded cartilage, which contained mostly clonal chondrocytes as confirmed by histology. The cartilage samples were ground in a freezer mill (Glen Creston, UK) and DNA was extracted with a Qiagen DNeasy maxi kit. To assess DNA methylation status, the genomic DNA was treated overnight with methylation-sensitive restriction enzymes. Cleavage of selected sites was detected by PCR amplifications with primer pairs designed to bracket selected promoter regions. Loss of the PCR band after digestion with the enzymes indicated absence of methylations, whereas presence of the band indicated methylated cytosine. We selected MMP-9 as one of genes that is activated in OA. Transcription of mmp-9 is regulated by a 670 bp sequence at the 5′-end flanking region, which contains 6 CpGs and a further 21 CpGs within the 1.5 kb region further upstream. A PCR primer pair was designed to bracket a 350bp sequence upstream from the transcription start site of mmp-9, which contained four of the six potential methylation sites, cleaved by the methylation-sensitive enzymes AciI and HhaI. DNA from 9 OA patients, 5 #NOF patients and 1 rheumatoid arthritic (RA) patient were digested with HhaI or AciI and examined for the presence or absence of PCR bands. In all patients, digestion with HhaI abolished the PCR band, indicating that the HhaI site was never methylated in either #NOF or OA patients. However, a remarkable difference was found after digestion with AciI: in 8/9 OA patients, the PCR band was no longer detectable, while in 4/5 #NOF patients the PCR band was still present. This suggested that all three AciI cleavage sites were methylated in the majority of chondrocytes from #NOF patients, while at least one of the three AciI cleavage sites was unmethylated in OA patients. Interestingly, the PCR band was present in the RA patient, suggesting methylation of the AciI cleavage sites. The present study provides the first ‘proof-of-concept’ data that suggest epigenetic changes may play a role in the etiology of osteoarthritis. Clearly further work is required to establish the generality of the present findings and whether de-methylations are also found in the promoter regions of other genes that are aberrantly expressed in OA.

Polysaccharide (alginate and chitosan) capsules coated with a unique self-assembled semi-crystalline shell of calcium phosphate provide an enclosed biological system for the spatial and temporal delivery of human cells and bioactive factors. The aim of this study was to demonstrate plasmid DNA entrapment, delivery and transfection of adjacent cells inside capsules, embedded capsules and plated. Bacterial plasmid DNA and/or bone cells (SaOS) was added to solution of sodium alginate solution supplemented with phosphate ions and mixed thoroughly. Alginate droplets were fed through a syringe into a solution of chitosan supplemented with calcium ions. Guest capsules were inserted into soft, pliable host capsules soon after immersion in chitosan solution. Capsules were then immersed in 2mL DMEM 10% FCS in 6-well plastic plates for up to 7 days to enable transfection to occur. Encapsulated bone cells were stained with standard X-Gal to show transfected cells expressing beta-galactosidase. DNA delivery and transfection was demonstrated within capsules containing SaOS cells and plasmid, an admixture of SaOS bone cells and plasmid (51%) and from capsules containing DNA alone suspended in media over plated SaOS one cells. We also demonstrate capsule transfection of encapsulated cells in vivo. Transfection efficiency is highest when plasmid is entrapped and released from embedded capsules followed by plasmid/ SaOS admixture within capsules and lowest efficiency was observed with plated SaOS cells (with a transfection efficiency of 5%). The ability to regulate shell decomposition by manipulating the degree of mineralization and the strength of gelling, and release of capsule contents provides a mechanism for programmed release of gene modulated cells into the biological environment. The beta-galactosidase plasmid was found to be strongly associated with the chitosan/ calcium phosphate shell as shown by ethidium-homodimer-1 staining of encapsulated DNA and this may assist the transfer from gel to cell. Programmed non-viral delivery of genes using biomaterial constructs is an important approach to gene therapy and orchestrated tissue regeneration. These unique biomineralised polysaccharide capsules provide a facile technique, and an enclosed biomimetic micro-environments with specifiable degradation characteristics, for the safe encapsulation and delivery of functional quantities of plasmid DNA with the implicit therapeutic implications therein.

Introduction: The ability to generate bone for skeletal repair, replacement or restoration is a major clinical need. Indeed the paucity of techniques in reconstructive surgery and trauma emphasise the need for alternative bone formation strategies. Natural biological ceramic structures possess arrangements of structural elements that govern and optimise tissue function, nutrition and organisation. The aim of this study was to fabricate biomineral microporous shells with highly complex forms and to examine their ability to interact with human osteoprogenitor cells as cell and growth factor delivery vehicles.

Methods: Microporous vaterite shells were generated using a synthetic in-solution mineralisation technique in which mineral is spontaneously deposited around vesicular templates (Walsh and Mann 1999)* Porous and textured self-organising hollow microspheres (5–20 _m) were generated expressing controlled and uniform shapes. These micropores puncture the surface at high densities and are interconnected throughout the sphere. Primary human bone marrow cells labelled with Cell Tracker Green and ethidium homodimer-1 fluorescent labels and osteoprogenitors transfected with an adenoviral vector expressing Green Fluorescent Protein (AdGFP) were cultured with vaterite shells over three weeks.

Results: Cell biocompatibility of these biomimetic spheres was confirmed by confocal fluorescence and light microscopy in primary human bone marrow cultures labelled with CTG and bone marrow cultures transfected with AdGFP. At three weeks microspheres were encapsulated and integrated with osteoprogenitor cells. Histological analysis confirmed expression of alkaline phosphatase, extracellular matrix synthesis and the capacity for extensive mineralisation. Examination by SEM, fluorescent and light microscopy showed that the growth of osteoprogenitors transfected with AdGFP and microspheres in pellet culture showed vaterite spheres were encapsulated and integrated within the osteoprogenitor cell matrix indicating the potential of growth factor delivery. To determine the potential of the spheres to encapsulate selected proteins, microporous spheres were incubated with bovine haemoglobin. FITC microscopic evidence showed haemoglobin could be entrapped inside the spheres and between the biomineral crystal plates during self-assembly.

Discussion and Conclusion: These studies demonstrate the development of facile techniques for the generation of porous microsphere sponges that are biocompatible, possess the ability to aid mineralisation and for the delivery of cell and growth factors. These calcium carbonate structures provide a material with widespread application in a range of tissue engineering applications including skeletal tissue regeneration.

Introduction: The clinical need for a biodegradable material with broad application is evidenced by the fact that tissue loss as a result of injury or disease provides reduced quality of life for many at significant socio-economic cost. The development of simple biodegradable materials, with broad applicability and tissue/ cell specificity has to date proved elusive. Natural biopolymers such as alginate and chitosan are structural biomaterials of increasing significance to tissue repair and regeneration due to their potential for fabrication, design and efficient, environmentally benign synthesis. We describe the development of innovative microcapsule scaffolds based on chitosan and alginate that can be tailored to a range of cell types for a variety of tissues.

Methods: Semi-permeable polysaccharide microcapsules were produced by a one-step method, in which the deposition of a semi-permeable alginate/chitosan membrane around droplets of sodium alginate was coupled with in-situ precipitation of amorphous calcium phosphate as described by Leveque et al (2002)*. A variety of human cell types including mesenchymal stem cells, osteoprogenitors selected using the STRO-1 antibody by magnetically activated cell separation (MACS), osteoprogenitors transfected with adenovirus expressing Green Fluorescent Protein (GFP) and chondrocytes were mixed with sodium alginate and encapsulated within alginate/chitosan and calcium phosphate.

Results: Hybrid spheres (750–10,000um) were generated encapsulating primary human osteoprogenitor cells, STRO-1 selected osteoprogenitors and AdGFP transfected osteoprogenitors. Encapsulated cells remain viable inside the polysaccharide microcapsules for 2 weeks as shown by positive alkaline phosphatase staining of encapsulated cells. Cells expressing GFP were observed within microspheres indicating the e ability to deliver cells/factors as well as the potential for gene therapy. Encapsulation and delivery of active BMP-2 was confirmed using the promyoblast cell line C2C12 known to be exquisitely sensitive to BMP-2. Nucleation of calcium phosphate occurred within the polysaccharide membrane and could be controlled by the phosphate concentration in the alginate droplets to produce hybrid microcapsules with enhanced mechanical strength. Thin walled capsules were shown to split and degrade in culture within 2–4 days releasing viable osteoprogenitor cells indicating the ability to manipulate the mechanical integrity and to programme degradation of the microspheres. Finally we have shown that aggregation of the microspheres into extended frameworks can be achieved using a designed droplet/vapour aerosol system resulting in foams of aggregated beads.

Discussion and Conclusion: A variety of human skeletal cells have been encapsulated within polysaccharide/ calcium phosphate microspheres and extended frameworks with specifiable dimensions. These composite scaffolds offer stable mechanical and chemical biomimetic environments conducive to normal cell function. Natural polysaccharides are also highly amenable to complexation with a range of bioactive molecules and consequently offer tremendous potential in tissue engineering and regeneration of hard and soft tissues.

Ex vivo gene transfer of osteogenic factors into multipotential stem cells offers potentially important therapeutic implications in a variety of musculoskeletal diseases. One possible approach is the development of a cellular vehicle, namely bone morphogenetic protein (BMP)-producing bone marrow cells, created using adenoviral gene transfer. These transduced cells provide local delivery of BMP for bone formation. The aims of this study were to study the feasibility of gene transfer to human bone osteoprogenitor cells, using adenoviral vectors. Specifically, the aims were to study the efficacy of transduction with an adenoviral vector expressing BMP-2 and then to determine the ability of the transduced cells to produce active BMP-2 and to generate bone ex vivo.

Primary human bone marrow osteoprogenitor cells were expanded in culture and infected with AxCALacZ, a replication-deficient adenoviral vector carrying the E. coli lacZ gene, with a range of multiplicity of infection (MOI) of 6.25 to100. Transduced cells showed positive staining for β-galactosidase using X-Gal with an efficiency close to 100%. Uninfected cells showed no β-galactosidase activity. Efficiency was independent from MOI, however cells infected at the lower MOIs expressed lower levels of β-galactosidase. Following confirmation that primary bone marrow cells could be infected by adenoviral constructs, additional osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene, at a multiplicity of infection of 10–20. In order to determine BMP-2 activity, conditioned media from bone marrow cells expressing BMP-2 was added to promyoblast C₂C₁₂ cells. The promyoblast C₂C₁₂ cells are exquisitely sensitive to BMP-2 with induction of alkaline phosphatase activity (ED₅₀ 20 nM) in a dose-dependant manner. Alkaline phosphatase activity was induced following culture with conditioned media from BMP-2 expressing cells, in a dose dependant manner, confirming successful secretion of active BMP-2. Immunohistochemical staining for alka- line phosphatase in C₂C₁₂ cells also confirmed the bio-chemical observations. Media from uninfected control human bone marrow cells failed to produce a similar effect. The concentration of BMP-2 in the media was estimated to be 5–10 nM/10⁷ cells.

To examine whether adenoviral transfection affected the osteoblast phenotype and their ability to mineralise in vitro, adenovirally-transduced bone marrow cells expressing BMP-2 were seeded onto poly(-lactic acid co÷glycolic acid) (75:25) porous scaffolds (provided by K. Shakesheff and S. Howdle; Nottingham University) and cultured for up to 6 weeks. Expression of alkaline phosphatase activity, type I collagen formation, as well as the synthesis of osteoblast stimulating factor-1 confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks.

These results indicate the ability to deliver active BMP-2 using human bone marrow osteoprogenitor cells following adenoviral infection. The maintenance of osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs offers a realistic approach to tissue engineer bone for orthopaedic applications.