Recent studies suggested that both the soluble protein of the mesenchymal stromal cell (MSC) secretome, as well as the secreted extracellular vesicles (EVs) promote bone regeneration. However, there is limited knowledge of the changes in MSC secretome vesicular fraction during aging. We therefore aimed to characterize the release profiles and cargo of EVs from MSCs of different chronological ages.

Conditioned medium (CM) was collected from 13 bone marrow MSC strains (20-89 years) and from one MSC strain derived from human induced pluripotent stem cells (iPSCs). The EV-containing fraction was enriched with ultracentrifugation. The number of particles in the CM was evaluated by nanoparticle tracking analysis (NTA), and the number of EVs was evaluated by flow cytometry (FC) after staining with cell-mask-green and anti-CD81 antibody. EV cargo analysis was conducted using next-generation sequencing (NGS).

Our data confirmed the release of EVs from all MSC strains used in the study. There were no correlations between the number of particles and the number of EVs released in the CM, and between the number of EVs released and the strain age. Nevertheless, some of the lowest concentrations of EVs were found in the CM of strains over 70 years of age, which exhibited a low/absent chondrogenic and osteogenic differentiation potential. In contrast, iPSC-MSCs, which exhibited a high growth and three-lineage differentiation potential, released a similar amount of EVs as the best performing bone marrow MSC strain. NGS analysis identified several microRNAs that were significantly enriched in EVs of young MSC strains exhibiting low senescence, and those that were enriched in EVs of strains exhibiting high differentiation potentials. Gender had no influence on microRNA profiles in EVs or releasing MSCs.

Taken together, our data provides new insights into the properties of MSC vesicular secretome and its therapeutic potential during aging.

Mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues in response to injury, such as fracture or other tissue injury. Bone marrow and adipose tissue are the major sources of MSCs. Previous studies suggested that the regenerative activity of stem cells can be enhanced by exposure to tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells-derived mesenchymal-like progenitors (hiPSCs-MPs) can enhance the regenerative potential of human bone marrow mesenchymal stromal cells (hBMSCs).

ECM was engineered from hiPSC-MPs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. hBMSCs were cultured on the engineered ECM, and differentiated into osteogenic, chondrogenic and adipogenic lineages. Growth and differentiation responses were compared to tissue culture plastic controls.

Decellularization of ECM resulted in efficient cell elimination, as observed in our previous studies. Cultivation hBMSCs on the ECM in osteogenic medium significantly increased hBMSC growth, collagen deposition and alkaline phosphatase activity. Furthermore, expression of osteogenic genes and matrix mineralization were significantly higher compared to plastic controls. Chondrogenic micromass culture on the ECM significantly increased cell growth and expression of chondrogenic markers, including glycosaminoglycans and collagen type II. Adipogenic differentiation of hBMSCs on the ECM resulted in significantly increased hBMSC growth, but significantly reduced lipid vacuole deposition compared to plastic controls. Together, our studies suggest that BMSCs differentiation into osteogenic and chondrogenic lineages can be enhanced, whereas adipogenic activity is decreased by the culture on engineered ECM. Contribution of specific matrix components and underlying mechanisms need to be further elucidated.

Our studies suggest that the three-lineage differentiation of aged BMSCs can be modulated by culture on hiPSC-engineered ECM. Further studies are aimed at scaling-up to three-dimensional ECM constructs for osteochondral tissue regeneration.

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs).

ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (<30 years) and aged (>70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic, as well as to collagen and fibronectin coated plates.

Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA, suggesting efficient cell elimination. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls and single protein substrates. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Engineered ECM induced a higher osteogenic response compared to specific matrix components.

Our studies suggest that aged BMSCs osteogenic activity can be enhanced by culture on engineered ECM. hiPSCs represent a scalable cell source, and tissue engineering strategies employing engineered ECM materials could potentially enhance bone regeneration in elderly patients.

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs).

ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic controls.

Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs.

Our studies suggest that aged BMSCs regenerative activity can be enhanced by culture on hiPSC-engineered ECM.

Due to unsatisfactory results and reported drawbacks of anterior cruciate ligament (ACL) reconstruction new regenerative approaches based on tissue-engineering strategies are currently under investigation. It was the purpose of this study to determine if a novel silk fiber-based ACL scaffold is able to initiate osteointegration in the femoral and tibial bone tunnels under in vivo conditions. Furthermore we tested if the osteointegration process will be improved by intraoperatively seeding the scaffolds with the autologous stromal vascular fraction, an adipose-derived, stem cell-rich isolate from knee fat pads.

In this controlled laboratory study, 33 sheep underwent ACL resection and were then randomly assigned to 2 experimental groups: ACL reconstruction with a scaffold alone and ACL reconstruction with a cell-seeded scaffold. Half of the sheep in each group were randomly chosen and euthanized 6 months after surgery and the other half at 12 months. To analyze the integration of the silk-based scaffold in the femoral and tibial bone tunnels, hard tissue histology and micro-computed tomography measurements were performed.

The histological workup showed that in all treatment groups, with or without the application of the autologous stromal vascular fraction, an interzone of collagen fibers had formed between bone and silk-based graft. This collagen-fiber continuity partly consisted of Sharpey fibers, comparable with tendon-bone healing known for autografts and allografts. Insertion sites were more broad based at 6 months and more concentrated on the slightly protruding, bony knoblike structures at 12 months. Histologically, no differences between the treatment groups were detectable. Analysis of micro-computed tomography measurements revealed a significantly higher tissue density for the cell-seeded scaffold group as compared with the scaffold-alone group in the tibial but not femoral bone tunnel after 12 months of implantation.

The novel silk fiber-based scaffold for ACL regeneration demonstrated integration into the bone tunnels via the formation of a fibrous interzone similar to allografts and autografts. Histologically, additional cell seeding did not enhance osteointegration. No significant differences between 6 and 12 months could be detected. After 12 months, there was still a considerable amount of silk present, and a longer observation period is necessary to see if a true ligament-bone enthesis will be formed.

The human amniotic membrane (hAM) contains cells of stem cell characteristics with low immunogenicity and anti-inflammatory properties and has for centuries been applied in the clinics especially for ophthalmology and wound care. It has recently been shown to be promising for novel applications such as tissue engineering and regenerative medicine. Towards these novel applications, we have demonstrated the potential of hAM in toto to differentiate towards bone, cartilage, Schwann like cells and recently also a producer of surfactant. We have further investigated the relevance of the location of origin for the therapeutic potential of the membrane. We show that placental and reflected hAM differs distinctly in morphology and functional activity. The placental region has significantly higher mitochondrial activity, however lower levels of reactive oxygen species, which suggests that placental and reflected regions may have different potential for tissue regeneration. We have further investigated the suitability of hAM to support therapeutic cells and have improved its maintenance in vitro towards xeno-free conditions.

Introduction: The present study was performed to compare the mechanical properties and fixation stability of tibial nails of the newest generation used in the management of distal metaphyseal fractures. Furthermore, we tried to evaluate whether distal locking with 4 locking screws might increase load-sharing after stabilization of distal metaphyseal tibial fractures.

Methods: We used 16 Sawbones third generation large left tibiae (Sawbones Inc., Sweden) to create an unstable distal metaphyseal fracture model (AO type 43-A3). In 8 specimens the fracture was stabilized with 2 nails with 3 distal locking options (4x VersaNail™, DePuy Orthopaedics, Johnson& Johnson, Warsaw, IN; 4x T2 Tibial Nailing System™, Stryker, Kiel, Germany) and in 8 specimens with 2 nails with 4 locking options (4x Connex™, ITS Spectromed, Lassnitzhöhe, Austria; 4x Expert Tibial Nail™, Synthes, Switzerland). Each specimen was loaded cyclically with three loading sequences over a period of 40,000 cycles in each series (700N, 1,500N, 1,800N). Implant stiffnes during axial cyclic loading series in 7° valgus alignement was recorded as well as cycles until failure of the bone-implant-construct.

Results: In the second loading series, implant failure was observed in all tibial nails with 3 distal locking screws after a mean period of 57,196.7 cycles. If distal locking was performed with 4 screws, implant failure was recorded in the third and last loading series after a mean period of 87,518.3 cycles (p< 0.001). If distal locking was performed with 3 distal locking screws, implant stiffness was 1776 (±99) N/mm. If distal locking was performed with 4 locking screws, implant stiffness was 2674 (±208) N/mm (p< 0.001).

Conclusion: Distal locking with 4 screws improves implant-bone stability. Stability is influenced by the number of locking screws and not by screw diameter. In these fracture type, nails with 4 distal locking options should be used.

We studied prospectively the regional inflammatory response to a unilateral distal radial fracture in 114 patients at eight to nine weeks after injury and again at one year. Our aim was to identify patients at risk for a delayed recovery and particularly those likely to develop complex regional pain syndrome. In order to quantify clinically the inflammatory response, a regional inflammatory score was developed. In addition, blood samples were collected from the antecubital veins of both arms for comparative biochemical and blood-gas analysis.

The severity of the inflammatory response was related to the type of treatment (Kruskal-Wallis test, p = 0.002). A highly significantly-positive correlation was found between the regional inflammatory score and the length of time to full recovery (r² = 0.92, p = 0.01, linear regession). A regional inflammatory score of 5 points with a sensitivity of 100% but a specificity of only 16% also identified patients at risk of complex regional pain syndrome. None of the biochemical parameters studied correlated with regional inflammatory score or predicted the development of complex regional pain syndrome.

Our study suggests that patients with a distal radial fracture and a regional inflammatory score of 5 points or more at eight to nine weeks after injury should be considered for specific anti-inflammatory treatment.

In fully-grown mongrel dogs, diaphyseal ulnar defects 25 mm long were stabilised by screws and plates, and were temporarily filled with silicone rubber blocks. After eight weeks the block was replaced either by fresh autogeneic cancellous bone, allogeneic deep-frozen cancellous bone, allogeneic decalcified bone matrix, or bone matrix gelatin. After 24 weeks the implants were evaluated by radiography, histology, and measurements of new bone volume, using computer-assisted density registration on microradiographs. Only the autogeneic bone grafts led to healing in all instances. Bone regeneration in the other groups was not significantly better than in the sham group in which no graft was employed. Decalcified bone matrix proved ineffective.