Intramedullary nailing of proximal humerus fractures does not achieve superior functional results to non-operative treatment in the long term.

INTRODUCTION: Non-operative treatment (NOT) of proximal humerus fractures (PHF) has regained significance due to recent evidence. Additionally, positive outcomes of plate osteosynthesis and fracture arthroplasty prompt a reassessment of the role of intramedullary nailing (IMN). While favorable short and medium-term results have been documented following IMN, little is known regarding functional outcomes and quality of life in the long-term. METHODS: Data from 180 patients with dislocated PHF of Neer types III, IV and V, treated at our level-I trauma center between 2004 and 2014 using IMN or NOT therapy, were scanned. Patients were re-evaluated after a minimum of 5 years to assess functional outcomes (age- and sex-adapted Constant Score, QuickDASH), quality of life (SF12), and complications or reoperations. RESULTS: Out of the initially identified 180 patients, 51 were unavailable for follow-up (FU) and 71 had deceased during the FU period. Functional outcomes and quality of life was, therefore, assessed in 58 patients (30 IMN, 28 NOT) with an average age at injury of 68 years after a mean FU time of 10.3 ± 3.4 years. Epidemiological patient characteristics did not exhibit significant differences between the two groups (p > .05). The functional outcome assessed by age- and sex-adapted Constant Score (NOT: 74 ± 28; IMN: 68 ± 24; p = .438), QuickDASH (NOT: 25 ± 27; IMN: 31 ± 23; p = .374) or quality of life using the SF12 (p > .05) revealed no significant disparities in long-term outcomes between the treatment groups. 10 of 30 patients in the IMN group underwent surgical revision to address complications, exceeding mere implant removal. Conversely, no patient in the NOT group underwent a revision surgery during the FU period. CONCLUSIONS: In the long-term, functional and quality of life-related outcomes of IMN did not diverge significantly from those of NOT, while causing a higher incidence of follow-up interventions.

Influence of preparation and football skill level on injury incidence during an amateur football tournament.

INTRODUCTION: Scientific studies on injury characteristics are rather common in professional football but not in amateur football despite the thousands of amateur football tournaments taking place worldwide each year. The purpose of this study was to evaluate the preparation and injury patterns of players of two different football skill levels who participated in an international amateur football tournament. METHODS: In a prospective cohort study, an international amateur football tournament of medical doctors in 2011 was analysed with regard to training and warm-up preparation, the level of football played before the tournament and injury data during the tournament by means of standardised injury definitions and data samples for football. RESULTS: Amateur players of registered football clubs had higher training exposure before the tournament (p < 0.001) than recreational players and had more frequently performed warm-up programmes (p < 0.001). Recreational football players showed a significantly higher overall injury incidence (p < 0.002), particularly of overuse injuries (p < 0.001), during the tournament than amateur players. In almost 75% of players in both groups, the body region most affected by injuries and complaints was the lower extremities. Orthopaedic and trauma surgeons had the lowest overall injury incidence and anaesthetists the highest (p = 0.049) during the tournament. CONCLUSION: For the first time, this study presents detailed information on the injury incidence and injury patterns of an amateur football tournament. Less-trained recreational players sustained significantly more injuries than better-trained amateur players, probably due to the lack of sufficient preparation before the tournament. Preventive strategies against overuse and traumatic injuries of recreational football players should start with regular training and warm-up programmes in preparation for a tournament.

RFE based chondroplasty in wrist arthroscopy indicates high risk for chrondocytes especially for the bipolar application.

BACKGROUND: The application of radiofrequency energy (RFE) has become widespread for surgical performed chondroplasty especially due to the anticipated sealing effect, however the safety of this procedure in the wrist remains unclear. The purpose of this study was to investigate the subchondral temperature during radiofrequency energy (RFE) application simulating chondroplasty in an arthroscopic setting of the wrist. METHODS: A chondroplasty of the lunate fossa was performed during an arthroscopy setting on 14 cadaver arms using monopolar or bipolar RFE. The temperature was recorded simultaneously from 7 predefined anatomical landmarks. RESULTS: The mean temperature for both application modes did not exceed more than 30°C at all measured points, except for the lunate fossa. The highest subchondral measured peak temperature was 49.35°C (monopolar) and 69.21°C (bipolar) in the lunate fossa. In addition, the temperature decreased for both radiofrequency (RF) devices depending on the distance of the sensors to the RF-probe. CONCLUSION: It remains to be questionable how safe RFE can be used for chondroplasty in wrist arthroscopy under continuous irrigation and constant movement to obtain the desired sealing effect. However, the bipolar device should be applied with more caution since peak temperature in the lunate fossa almost reached 70°C even under continuous irrigation.

Screw placement in percutaneous acetabular surgery: gender differences of anatomical landmarks in a cadaveric study.

PURPOSE: Percutaneous reduction and periarticular screw implantation techniques have been successfully introduced in acetabular surgery. The advantages of this less invasive approach are attenuated by higher risks of screw misplacement. Anatomical landmarks are strongly needed to prevent malplacement. This cadaver study was designed to identify reliable anatomical osseous landmarks in the pelvic region for screw placement in acetabular surgery. Gender differences were specifically addressed. METHODS: Twenty-seven embalmed cadaveric hemipelvic specimens (13 male, 14 female) were used. After soft-tissue removal, anterior and posterior column acetabular screw placement was conducted by one orthopaedic trauma surgeon under direct vision. Each column was addressed by antegrade and retrograde screw insertion. Radiographic verification of ideal screw placement was followed by assessment of the distance from the different entry points to adjoining anatomical osseous structures. RESULTS: For anterior column screw positioning, the posterior superior iliac spine (PSIS), posterior inferior iliac spine (PIIS), iliopectineal eminence and centre of the symphysis were most reliable regarding gender differences. For posterior column screw positioning, the distance to the anterior superior iliac spine (ASIS) and the ischial tuberosity showed the lowest deviation between the different gender specimens. Highest gender differences were seen in relation to the cranial rim of the superior pubic ramus in retrograde anterior column screw positioning (p = 0.002). Most landmarks could be targeted within a 2.5-cm range in all specimens. CONCLUSIONS: The findings emphasise the relevance of osseous landmarks in acetabular surgery. By adhering to easily identifiable structures, screw placement can be safely performed. Significant gender differences must be taken into consideration during preoperative planning.

Effect of parathyroid hormone-related protein in an in vitro hypertrophy model for mesenchymal stem cell chondrogenesis.

PURPOSE: Mesenchymal stem cells (MSCs) express markers of hypertrophic chondrocytes during chondrogenic differentiation. We tested the suitability of parathyroid hormone-related protein (PTHrP), a regulator of chondrocyte hypertrophy in embryonic cartilage development, for the suppression of hypertrophy in an in vitro hypertrophy model of chondrifying MSCs. METHODS: Chondrogenesis was induced in human MSCs in pellet culture for two weeks and for an additional two weeks cultures were either maintained in standard chondrogenic medium or transferred to a hypertrophy-enhancing medium. PTHrP(1-40) was added to the medium throughout the culture period at concentrations from 1 to 1,000 pM. Pellets were harvested on days one, 14 and 28 for biochemical and histological analysis. RESULTS: Hypertrophic medium clearly enhanced the hypertrophic phenotype, with increased cell size, and strong alkaline phosphatase (ALP) and type X collagen staining. In chondrogenic medium, 1-100 pM PTHrP(1-40) did not inhibit chondrogenic differentiation, whereas 1,000 pM PTHrP(1-40) significantly reduced chondrogenesis. ALP activity was dose-dependently reduced by PTHrP(1-40) at 10-1,000 pM in chondrogenic conditions. Under hypertrophy-enhancing conditions, PTHrP(1-40) did not inhibit the induction of the hypertrophy. At the highest concentration (1,000 pM) in the hypertrophic group, aggregates were partially dedifferentiated and differentiated areas of these aggregates maintained their hypertrophic appearance. CONCLUSIONS: PTHrP(1-40) treatment dose-dependently reduced ALP expression in MSC pellets cultured under standard chondrogenic conditions and is thus beneficial for the maintenance of the chondrogenic phenotype in this medium condition. When cultured under hypertrophy-enhancing conditions, PTHrP(1-40) could not diminish the induced enhancement of hypertrophy in the MSC pellets.

[Humeral bone defects in revision shoulder arthroplasty]. / Humerale Knochendefekte in der Revisionsendoprothetik.

BACKGROUND: Revision shoulder arthroplasty is mainly performed with reverse TSA and should consider proper adjustment of the length and the amount of bone loss in humeral reconstruction. Whilst epi-/metaphyseal bone loss can mostly be compensated easily by stemmed standard implants, advanced bone loss exceeding 2° requires the support of longer revision stems. EXTENSIVE HUMERAL BONE LOSS: Cementless fixation in the intact diaphyseal humerus is recommended in bone loss exceeding 2°, preferably with modular revision systems, because cemented reverse revision stems have higher loosening rates in the mid to long-term follow-up. In cases of advanced bone loss 3°-4° (more than 6-7 cm), structural humeral allografts should be considered to prevent instability and early loosening. Unfortunately, the access to fresh frozen allografts is very limited due to regulation of the German government in contrast to the situation in the US or Switzerland. Reverse tumor arthroplasty is an option with a higher complication rate and inferior function even when polyester mesh is used for ingrowth of soft tissues. DISTINCT DIAPHYSEAL DEFECTS: In bone loss 4°-5° the minimal anchorage length is mostly critical due to the curvature of the medullary canal. The fixation of a revision stem is only possible when at least 2-3 widths of the diaphyseal diameter are available. Custom-made implants with flanges or distal locking screws, or bipolar tumor arthroplasty may be required. Additionally, strut allografts can be useful to achieve stable fixation. Two-stage biological reconstruction in impaction-bone-graft or the Masquelet technique are rarely used as a salvage procedure.

Predictors of Mortality in Head-Preserving Treatment for Dislocated Proximal Humerus Fractures: A Retrospective Analysis of 522 Cases with a Minimum Follow-Up of 5 Years.

PURPOSE: Proximal humerus fractures (PHFs) are among the most common fractures in elderly patients, but there is still inadequate knowledge about mortality risk factors after such injuries. In order to provide the best possible therapy, individual risk factors have to be considered and evaluated thoroughly. There is still controversy regarding treatment decisions for proximal humerus fractures, particularly for the elderly. METHODS: In this study, patient data from 522 patients with proximal humerus fractures were obtained from 2004 to 2014 at a Level 1 trauma centre. After a minimum follow-up of 5 years, the mortality rate was assessed, and independent risk factors were evaluated. RESULTS: A total of 383 patients (out of 522) were included in this study. For our patient collective, the mean follow-up was at 10.5 ± 3.2 years. The overall mortality rate was 43.8% in our respondent group and was not significantly impacted by concomitant injuries. The binary logistic regression model showed an increased risk for mortality by 10% per life year, a 3.9 times higher mortality risk for men and a 3.4 times higher risk for conservative treatment. The most powerful predictor was a Charlson Comorbidity Index of more than 2, with a 20 times higher mortality risk. CONCLUSIONS: Outstanding independent predictors of death in our patient collective were serious comorbidities, male patients, and conservative treatment. This patient-related information should influence the process of decision making for the individual treatment of patients with PHFs.

Treatment of long bone defects and non-unions: from research to clinical practice.

The treatment of long bone defects and non-unions is still a major clinical and socio-economical problem. In addition to the non-operative therapeutic options, such as the application of various forms of electricity, extracorporeal shock wave therapy and ultrasound therapy, which are still in clinical use, several operative treatment methods are available. No consensus guidelines are available and the treatments of such defects differ greatly. Therefore, clinicians and researchers are presently investigating ways to treat large bone defects based on tissue engineering approaches. Tissue engineering strategies for bone regeneration seem to be a promising option in regenerative medicine. Several in vitro and in vivo studies in small and large animal models have been conducted to establish the efficiency of various tissue engineering approaches. Neverthelsss, the literature still lacks controlled studies that compare the different clinical treatment strategies currently in use. However, based on the results obtained so far in diverse animal studies, bone tissue engineering approaches need further validation in more clinically relevant animal models and in clinical pilot studies for the translation of bone tissue engineering approaches into clinical practice.

Cartilage labelling for mechanical testing in T-peel configuration.

PURPOSE: The purpose of this study was to find a suitable method of labelling cartilage samples for the measurement of distraction distances in biomechanical testing. METHODS: Samples of bovine cartilage were labelled using five different methods: hydroquinone and silver nitrate (AgNO3), potassium permanganate (KMnO4) with sodium thiosulphate (Na2S2O3), India ink, heat, and laser energy. After the labelling, we analysed the cartilage samples with regard to cytotoxity by histochemical staining with ethidiumbromide homodimer (EthD-1) and calcein AM. Furthermore, we tested cartilages labelled with India ink and heat in a T-peel test configuration to analyse possible changes in the mechanical behaviour between marked and unlabelled samples. RESULTS: Only the labelling methods with Indian ink or a heated needle showed acceptable results in the cytotoxity test with regard to labelling persistence, accuracy, and the influence on consistency and viability of the chondrocytes. In the biomechanical T-peel configuration, heat-labelled samples collapsed significantly earlier than unlabelled samples. CONCLUSION: Labelling bovine cartilage samples with Indian ink in biomechanical testing is a reliable, accurate, inexpensive, and easy-to-perform method. This labelling method influenced neither the biomechanical behaviour nor the viability of the tissue compared to untreated bovine cartilage.

A minimally invasive stabilizing system for dorsal pelvic ring injuries.

BACKGROUND: Open reduction and stabilization of dorsal pelvic ring injuries is accompanied by a high rate of soft tissue complications. Minimally invasive techniques have the potential to decrease soft tissue trauma, but the risk of iatrogenic nerve and vessel damage through the reduced surgical exposure should be considered. We treated these injuries using a transiliac internal fixator (TIFI) in a minimally invasive technique characterized by implantation of a pedicle screw and rod system, bridging the sacroiliac joints and the sacral area. QUESTIONS/PURPOSES: We asked whether (1) we could achieve anatomic restoration with the device, (2) specific complications were associated with this minimally invasive approach (particularly enhanced intraoperative blood loss, soft tissue complications, and iatrogenic neurovascular damage), and (3) function 3 years after trauma was comparable to that of established methods. METHODS: We retrospectively reviewed 67 patients with dorsal pelvic injuries during a 7-year period. We evaluated the (1) reduction by grading the maximal displacement measured with three radiographic views, (2) the complications during the observation period, and (3) the function with a validated questionnaire (Pelvic Outcome Score) in all but five patients at least 3 years after trauma (mean, 37 months; range, 36-42 months). RESULTS: At last followup we observed a secondary fracture displacement greater than 5 mm in one patient. The intraoperative blood loss was less than 50 mL in all patients. No neurovascular lesions occurred owing to implantation. Four patients had wound infections, one had loosening of a single pedicle screw, and one had an iatrogenic screw malpositioning. Thirty-five of the 62 patients achieved Pelvic Outcome Scores of either a maximum score or 6 of 7 points. CONCLUSION: Our observations suggest TIFI is a reasonable alternative to other established fixation devices for injuries of the dorsal pelvic ring with minor risks of major blood loss or iatrogenic neurovascular damage. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Biomechanical analysis of a transiliac internal fixator.

PURPOSE: We evaluated the biomechanical characteristics of the transiliac internal fixator (TIFI) as compared to two well-established methods of internal posterior pelvic ring fixation. METHODS: Six freshly frozen human pelves were used for simulated single-leg stance loading of an AO type C injury model (pubic symphysis diastasis and unilateral sacroiliac joint disruption). The symphysis rupture was stabilized with a dynamic compression plate. Afterwards the three internal stabilization systems (TIFI, iliosacral screws and ventral plate osteosynthesis) were analysed. Fragment movement was measured in a contact-free manner with a stereophotometric infrared system. RESULTS: No significant differences in the three-dimensional deformation tolerated by the TIFI as compared to the other internal fixation systems were found. CONCLUSIONS: The transiliac internal fixator provides the same biomechanical stability as the other reference implants tested. We suggest the use of this device as a suitable alternative to the other implants.

Arthroplasty of the lunate using bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells have the potential to differentiate into a variety of mesenchymal tissues such as bone, cartilage and ligaments. The potential for the regeneration of bone with cartilage coverage has still not been achieved. We evaluated the ability of bone marrow mesenchymal stromal cells to regenerate osteochondral defects in the cavity of the lunate in an animal model. Autologous mesenchymal stromal cells were harvested from the iliac crest of New Zealand white rabbits and expanded in vitro. Total lunate excision was performed in 24 animals and the isolated cells were loaded onto scaffolds. Cell-free scaffolds were implanted in the lunate space of the right wrists of all animals, and the left lunate spaces were filled with predifferentiated, cell-loaded scaffolds. Radiographic and histological analyses were performed after two, six and 12 weeks. In addition, the animals were injected with a fluorescent agent every five days, starting at day 30. After two and six weeks there was no radiographic evidence of ossification, whereas after 12 weeks all animals showed radiographic evidence of ossification. Histological sections showed increasing evidence of cartilage-like cell formation at the edges and new bone tissue in the centre of the newly formed tissue in all groups. The histological examinations showed that bone tissue was located around the newly incorporated vascularisation. This study demonstrated that newly formed vascularisation is necessary for the regeneration of bone tissue with cell-loaded scaffolds.

Scaffold-guided bone regeneration in large volume tibial segmental defects.

Large volume losses in weight bearing long bones are a major challenge in clinical practice. Despite multiple innovations over the last decades, significant limitations subsist in current clinical treatment options which is driving a strong clinical demand for clinically translatable treatment alternatives, including bone tissue engineering applications. Despite these shortcomings, preclinical large animal models of large volume segmental bone defects to investigate the regenerative capacity of bone tissue engineering strategies under clinically relevant conditions are rarely described in literature. We herein present a newly established preclinical ovine animal model for the treatment of XL volume (19 cm3) segmental tibial defects. In eight aged male Merino sheep (age > 6 years) a mid-diaphyseal tibial segmental defect was created and stabilized with a 5.6 mm Dynamic Compression Plate (DCP). We present short-term (3 months) and long-term (12-15 months) results of a pilot study using medical grade Polycaprolactone-Tricalciumphosphate (mPCL-TCP) scaffolds combined with a dose of 2 mg rhBMP-7 delivered in Platelet-Rich- Plasma (PRP). Furthermore, detailed analyses of the mechanical properties of the scaffolds as well as interfragmentary movement (IFM) and DCP-surface strain in vitro and a comprehensive description of the surgical and post-surgery protocol and post-mortem analysis is given.

Hypertrophy in mesenchymal stem cell chondrogenesis: effect of TGF-beta isoforms and chondrogenic conditioning.

Induction of chondrogenesis in mesenchymal stem cells (MSCs) with TGF-beta leads to a hypertrophic phenotype. The hypertrophic maturation of the chondrocytes is dependent on the timed removal of TGF-beta and sensitive to hypertrophy-promoting agents in vitro. In this study, we have investigated whether TGF-beta3, which has been shown to be more prochondrogenic compared to TGF-beta1, similarly enhances terminal differentiation in an in vitro hypertrophy model of chondrogenically differentiating MSCs. In addition, we tested the impact of the time of chondrogenic conditioning on the enhancement of hypertrophy. MSCs were chondrogenically differentiated in pellet culture in medium containing TGF-beta1 or TGF-beta3. After 2 or 4 weeks, chondrogenic medium was switched to hypertrophy-inducing medium for 2 weeks. Aggregates were analyzed histologically and biochemically on days 14, 28 and 42. The switch to hypertrophy medium after 14 days induced hypertrophic cell morphology and significant increase in alkaline phosphatase activity compared to the chondrogenesis only control using both TGF-beta1 and TGF-beta3. After 28 days predifferentiation, differences between hypertrophic and control groups diminished compared to 14 days predifferentiation. In conclusion, chondrogenic conditioning with both TGF-beta isoforms similarly induced hypertrophy in our experiment and allowed the enhancement of the hypertrophic chondrocyte phenotype by hypertrophic medium. Enhancement of hypertrophy was seen more clearly after the shorter chondrogenic conditioning. Therefore, to utilize this experimental model as a tool to study hypertrophy in MSC chondrogenesis, a predifferentiation period of 14 days is recommended.

A preclinical large-animal model for the assessment of critical-size load-bearing bone defect reconstruction.

Critical-size bone defects, which require large-volume tissue reconstruction, remain a clinical challenge. Bone engineering has the potential to provide new treatment concepts, yet clinical translation requires anatomically and physiologically relevant preclinical models. The ovine critical-size long-bone defect model has been validated in numerous studies as a preclinical tool for evaluating both conventional and novel bone-engineering concepts. With sufficient training and experience in large-animal studies, it is a technically feasible procedure with a high level of reproducibility when appropriate preoperative and postoperative management protocols are followed. The model can be established by following a procedure that includes the following stages: (i) preoperative planning and preparation, (ii) the surgical approach, (iii) postoperative management, and (iv) postmortem analysis. Using this model, full results for peer-reviewed publication can be attained within 2 years. In this protocol, we comprehensively describe how to establish proficiency using the preclinical model for the evaluation of a range of bone defect reconstruction options.

TGF-ß Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-ß Receptor Downregulation.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) become hypertrophic in long term despite chondrogenic differentiation following the pathway of growth plate chondrocytes. This terminal differentiation leads to phenotypically unstable cartilage and was mirrored in vitro by addition of hypertrophy inducing medium. We investigated how intrinsic TGF-ß signaling is altered in pro-hypertrophic conditions. METHODS AND RESULTS: Human bone marrow derived MSC were chondrogenically differentiated in 3D culture. At day 14 medium conditions were changed to 1. pro-hypertrophic by addition of T3 and withdrawal of TGF-ß and dexamethasone 2. pro-hypertrophic by addition of BMP 4 and withdrawal of TGF-ß and dexamethasone and 3. kept in prochondrogenic medium conditions. All groups were treated with and without TGFß-type-1-receptor inhibitor SB431542 from day 14 on. Aggregates were harvested for histo- and immunohistological analysis at d14 and d28, for gene expression analysis (rt-PCR) on d1, d3, d7, d14, d17, d21 and d28 and for Western blot analysis on d21 and d28. Induction of hypertrophy was achieved in the pro-hypertrophic groups while expression of TGFß-type-1- and 2-receptor and Sox 9 were significantly downregulated compared to pro-chondrogenic conditions. Western blotting showed reduced phosphorylation of Smad 2 and 3 in hypertrophic samples, reduced TGF-ß-1 receptor proteins and reduced SOX 9. Addition of SB431542 did not initiate hypertrophy under pro-chondrogenic conditions, but was capable of enhancing hypertrophy when applied simultaneously with BMP-4. CONCLUSIONS: Our results suggest that the enhancement of hypertrophy in this model is a result of both activation of pro-hypertrophic BMP signaling and reduction of anti-hypertrophic TGFß signaling.

Poly(ε-caprolactone) Scaffolds Fabricated by Melt Electrospinning for Bone Tissue Engineering.

Melt electrospinning is a promising approach to manufacture biocompatible scaffolds for tissue engineering. In this study, melt electrospinning of poly(ε-caprolactone) onto structured, metallic collectors resulted in scaffolds with an average pore size of 250-300 µm and an average fibre diameter of 15 µm. Scaffolds were seeded with ovine osteoblasts in vitro. Cell proliferation and deposition of mineralised extracellular matrix was assessed using PicoGreen® (Thermo Fisher Scientific, Scoresby, Australia) and WAKO® HR II (WAKO, Osaka, Japan) calcium assays. Biocompatibility, cell infiltration and the growth pattern of osteoblasts on scaffolds was investigated using confocal microscopy and scanning electron microscopy. Osteoblasts proliferated on the scaffolds over an entire 40-day culture period, with excellent survival rates and deposited mineralized extracellular matrix. In general, the 3D environment of the structured melt electrospun scaffold was favourable for osteoblast cultures.

Higher Ratios of Hyaluronic Acid Enhance Chondrogenic Differentiation of Human MSCs in a Hyaluronic Acid-Gelatin Composite Scaffold.

Mesenchymal stem cells (MSCs) seeded on specific carrier materials are a promising source for the repair of traumatic cartilage injuries. The best supportive carrier material has not yet been determined. As natural components of cartilage's extracellular matrix, hyaluronic acid and collagen are the focus of biomaterial research. In order to optimize chondrogenic support, we investigated three different scaffold compositions of a hyaluronic acid (HA)-gelatin based biomaterial. METHODS: Human MSCs (hMSCs) were seeded under vacuum on composite scaffolds of three different HA-gelatin ratios and cultured in chondrogenic medium for 21 days. Cell-scaffold constructs were assessed at different time points for cell viability, gene expression patterns, production of cartilage-specific extracellular matrix (ECM) and for (immuno-)histological appearance. The intrinsic transforming growth factor beta (TGF-beta) uptake of empty scaffolds was evaluated by determination of the TGF-beta concentrations in the medium over time. RESULTS: No significant differences were found for cell seeding densities and cell viability. hMSCs seeded on scaffolds with higher ratios of HA showed better cartilage-like differentiation in all evaluated parameters. TGF-beta uptake did not differ between empty scaffolds. CONCLUSION: Higher ratios of HA support the chondrogenic differentiation of hMSCs seeded on a HA-gelatin composite scaffold.

Expression of BMP and Actin Membrane Bound Inhibitor Is Increased during Terminal Differentiation of MSCs.

Chondrogenic differentiating mesenchymal stem cells (MSCs) are mimicking embryonal endochondral ossification and become hypertrophic. BMP (bone morphogenetic protein) and Activin Membrane Bound Inhibitor (BAMBI) is a pseudoreceptor that regulates the activity of transforming growth factor-ß (TGF-ß) and BMP signalling during chondrogenesis. Both TGF-ß and BMP signalling are regulators of chondrogenic cell differentiation. Human bone marrow derived MSCs were chondrogenically predifferentiated in aggregate culture for 14 days. Thereafter, one group was subjected to hypertrophy enhancing media conditions while controls were kept in chondrogenic medium until day 28. Histological evaluation, gene expression by PCR, and Western blot analysis were carried out at days 1, 3, 7, 14, 17, 21, and 28. A subset of cultures was treated with the BMP inhibitor Noggin to test for BMP dependent expression of BAMBI. Hypertrophic differentiated pellets showed larger cells with increased collagen 10 and alkaline phosphatase staining. There was significantly increased expression of BAMBI on gene expression and protein level in hypertrophic cultures compared to the chondrogenic control and increased BMP4 gene expression. Immunohistochemistry showed intense staining of BAMBI in hypertrophic cells. BAMBI expression was dose-dependently downregulated by Noggin. The pseudoreceptor BAMBI is upregulated upon enhancement of hypertrophy in MSC chondrogenic differentiation by a BMP dependent mechanism.

Microparticles for Sustained Growth Factor Delivery in the Regeneration of Critically-Sized Segmental Tibial Bone Defects.

This study trialled the controlled delivery of growth factors within a biodegradable scaffold in a large segmental bone defect model. We hypothesised that co-delivery of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) followed by bone morphogenetic protein-2 (BMP-2) could be more effective in stimulating bone repair than the delivery of BMP-2 alone. Poly(lactic-co-glycolic acid) (PLGA ) based microparticles were used as a delivery system to achieve a controlled release of growth factors within a medical-grade Polycaprolactone (PCL) scaffold. The scaffolds were assessed in a well-established preclinical ovine tibial segmental defect measuring 3 cm. After six months, mechanical properties and bone tissue regeneration were assessed. Mineralised bone bridging of the defect was enhanced in growth factor treated groups. The inclusion of VEGF and PDGF (with BMP-2) had no significant effect on the amount of bone regeneration at the six-month time point in comparison to BMP-2 alone. However, regions treated with VEGF and PDGF showed increased vascularity. This study demonstrates an effective method for the controlled delivery of therapeutic growth factors in vivo, using microparticles.