Development of a clinically relevant strategy to promote fracture healing in an atrophic non-union model using mesenchymal stem cells

Atrophic non-union is a major complication following fracture of a bone. It represents a biological failure of the fracture healing process and occurs in 5-10% of cases. A number of factors predispose to atrophic non-union including high energy injuries, open fractures, diabetes, and smoking. Atrophic non-unions cause immense patient morbidity and consume large amount of health care resources. Bone grafts taken from the iliac crest contain biologic components required for fracture healing and are considered as the gold standard treatment of aseptic atrophic non-union. However, harvesting bone grafts from the iliac crest is associated with significant patient morbidity which can reduce quality of life. Mesenchymal stem cells (MSCs) have the ability to proliferate and undergo multilineage differentiation. The emergence of MSC therapy provides an alternative strategy for treating impaired fracture healing. MSCs contribute to normal fracture healing both directly as bone progenitor cells and indirectly as mediator secreting cells. Although a number of studies have shown that MSCs can promote bone regeneration in small animal fresh critical size defects, this is not analogous to most clinical aseptic atrophic non-unions which do not have a significant bone gap. There remains therefore a clinical need for an appropriate strategy for using stem cells in atrophic non-unions. Thus, the aim of this study aim was to develop a clinically relevant strategy to promote fracture healing in an atrophic non-union model using the percutaneous injection of MSCs as a minimally invasive technique. An atrophic non-union model was established and validated. A small (1 mm) non-critical size defect was created at the mid shaft tibia and the fracture site was stabilised using an external fixator. Atrophic non-union was induced by stripping the periosteum for one bone diameter either side of the osteotomy site and curettage of the intramedullary canal over the same distance. The procedure reliably created an atrophic non-union. Fracture healing was evaluated using (1) serial radiography, (2) micro-computed tomography, (3) histomorphology and (5) biomechanical testing. Fracture scoring systems including the radiographic union scale in tibia (RUST) and the Lane & Sandhu score were validated in a preclinical model. A simple sample preparation technique for evaluating bone mechanical properties was developed and used to assess the stiffness and strength of the fracture repair. Percutaneous injection of MSCs locally into the fracture site in the early ‘post-injury’ period at three weeks after induction of atrophic non-union was found to improve the fracture healing process significantly (83% of cases), while MSCs implantation in the late ‘post-injury’ period at eight weeks after induction of atrophic non-union showed no significant improvement of fracture healing (20% of cases). Percutaneous local implantation of MSCs rescued the fracture healing process in cases destined to progress to atrophic non-union. In clinical practice, there may be an advantage using MSCs from a universal donor as the processes of MSC isolation and preparation are expensive and time consuming. To investigate the feasibility of using non-autologous cells, the atrophic non-union was used to determine the bone regenerative potential of using xenogeneic donor hMSCs in an atrophic non-union. The results demonstrated that the therapeutic effect of using hMSCs in a xenogeneic manner to promote fracture healing in the rat atrophic non-union model was comparable with rMSCs (88% of cases in both hMSCs and rMSCs) and there were neither significant clinical adverse effects nor adverse immune responses with the xenogeneic transplantation. However, MSCs did not persist at the fracture following injection. Perivascular stem cells (PSCs) taken from adipose tissue, which is an expendable source, have advantages over conventional MSCs as they are a defined and homogenous population and can be used without culture expansion. The administration of PSC using percutaneous injection improved the fracture healing process in atrophic non-union (60% of cases). This suggested that PSCs may present an appropriate choice for use in cell therapies to promote fracture healing in atrophic non-union. The results from this thesis can be applied to the development of a clinically relevant strategy using MSCs as a minimally invasive technique to promote fracture healing in atrophic non-union, in particular (1) the effectiveness of a cell therapy is likely to be highly dependent of the timing of injection relative to the stage of fracture healing, (2) hMSCs were as effective as rMSCs in promoting fracture healing, suggesting that it may be feasible to use an allogeneic strategy in humans, (3) the injected MSCs were not detectable even in case of successful repair, suggesting that they may act through a paracrine effect and (4) PSCs isolated from adipose tissue contributed to fracture healing in the atrophic non-union model, suggesting that adipose tissues can be used as an alternative cell sources for bone repair

A genetic association study between growth differentiation factor 5 (GDF 5) polymorphism and knee osteoarthritis in Thai population

Abstract Objective Osteoarthritis (OA) is a multi-factorial disease and genetic factor is one of the important etiologic risk factors. Various genetic polymorphisms have been elucidated that they might be associated with OA. Recently, several studies have shown an association between Growth Differentiation Factor 5(GDF5) polymorphism and knee OA. However, the role of genetic predisposing factor in each ethnic group cannot be replicated to all, with conflicting data in the literatures. Therefore, the aim of this study was to investigate the association between GDF5 polymorphism and knee OA in Thai population. Materials and Methods One hundred and ninety three patients aged 54-88 years who attended Ramathibodi Hospital were enrolled. Ninety cases with knee OA according to American College of Rheumatology criteria and one hundred and three cases in control group gave informed consent. Blood sample (5 ml) were collected for identification of GDF5 (rs143383) single nucleotide polymorphism by PCR/RFLP according to a standard protocol. This study protocol was approved by the Ethics Committee on human experimentation of Ramathibodi Hospital Faculty of Medicine, Mahidol University. Odds ratios (OR) and 95% confidence intervals were calculated for the risk of knee OA by genotype (TT, TC and CC) and allele (T/C) analyses. Results The baseline characteristics between two groups including job, smoking and activity were not different, except age and BMI. The entire cases and controls were in Hardy-Weinberg equilibrium (p > 0.05). The OA knee group (n = 90) had genotypic figure which has shown by TT 42.2% (n = 38), TC 45.6% (n = 41) and CC 12% (n = 11), whereas the control group (n = 103) revealed TT 32% (n = 33), TC 45.6% (n = 47), and CC 22.3% (n = 23), respectively. Genotypic TT increased risk of knee OA as compared to CC [OR = 2.41 (P = 0.04, 95%CI = 1.02-5.67)]. In the allele analysis, the T allele was found to be significantly associated with knee OA [OR = 1.53 (P = 0.043, 95%CI = 1.01-2.30)]. Conclusion These data suggested that GDF5 polymorphism has an association with knee OA in Thai ethnic. This finding also supports the hypothesis that OA has an important genetic component in its etiology, and GDF5 protein might play important role in the pathophysiology of the disease.</p

Infrapatellar Fat Pad:An Alternative Source of Adipose-Derived Mesenchymal Stem Cells

Introduction. The Infrapatellar fat pad (IPFP) represents an emerging alternative source of adipose-derived mesenchymal stem cells (ASCs). We compared the characteristics and differentiation capacity of ASCs isolated from IPFP and SC. Materials and Methods. ASCs were harvested from either IPFP or SC. IPFPs were collected from patients undergoing total knee arthroplasty (TKA), whereas subcutaneous tissues were collected from patients undergoing lipoaspiration. Immunophenotypes of surface antigens were evaluated. Their ability to form colony-forming units (CFUs) and their differentiation potential were determined. The ASCs karyotype was evaluated. Results. There was no difference in the number of CFUs and size of CFUs between IPFP and SC sources. ASCs isolated from both sources had a normal karyotype. The mesenchymal stem cells (MSCs) markers on flow cytometry was equivalent. IPFP-ASCs demonstrated significantly higher expression of SOX-9 and RUNX-2 over ASCs isolated from SC (6.19 ± 5.56-, 0.47 ± 0.62-fold; p value = 0.047, and 17.33 ± 10.80-, 1.56 ± 1.31-fold; p value = 0.030, resp.). Discussion and Conclusion. CFU assay of IPFP-ASCs and SC-ASCs harvested by lipoaspiration technique was equivalent. The expression of key chondrogenic and osteogenic genes was increased in cells isolated from IPFP. IPFP should be considered a high quality alternative source of ASCs

The radiographic union scale in tibial (RUST) fractures:Reliability of the outcome measure at an independent centre

OBJECTIVES: The radiographic union score for tibial (RUST) fractures was developed by Whelan et al to assess the healing of tibial fractures following intramedullary nailing. In the current study, the repeatability and reliability of the RUST score was evaluated in an independent centre (a) using the original description, (b) after further interpretation of the description of the score, and (c) with the immediate post-operative radiograph available for comparison. METHODS: A total of 15 radiographs of tibial shaft fractures treated by intramedullary nailing (IM) were scored by three observers using the RUST system. Following discussion on how the criteria of the RUST system should be implemented, 45 sets (i.e. AP and lateral) of radiographs of IM nailed tibial fractures were scored by five observers. Finally, these 45 sets of radiographs were rescored with the baseline post-operative radiograph available for comparison. RESULTS: The initial intraclass correlation (ICC) on the first 15 sets of radiographs was 0.67 (95% CI 0.63 to 0.71). However, the original description was being interpreted in different ways. After agreeing on the interpretation, the ICC on the second cohort improved to 0.75. The ICC improved even further to 0.79, when the baseline post-operative radiographs were available for comparison. CONCLUSION: This study demonstrates that the RUST scoring system is a reliable and repeatable outcome measure for assessing tibial fracture healing. Further improvement in the reliability of the scoring system can be obtained if the radiographs are compared with the baseline post-operative radiographs. Cite this article: Mr J.M. Leow. The radiographic union scale in tibial (RUST) fractures: Reliability of the outcome measure at an independent centre. Bone Joint Res 2016;5:116–121. DOI: 10.1302/2046-3758.54.2000628

Isolation and characterization of equine native MSC populations

Abstract Background In contrast to humans in which mesenchymal stem/stromal cell (MSC) therapies are still largely in the clinical trial phase, MSCs have been used therapeutically in horses for over 15 years, thus constituting a valuable preclinical model for humans. In human tissues, MSCs have been shown to originate from perivascular cells, namely pericytes and adventitial cells, which are identified by the presence of the cell surface markers CD146 and CD34, respectively. In contrast, the origin of MSCs in equine tissues has not been established, preventing the isolation and culture of defined cell populations in that species. Moreover, a comparison between perivascular CD146+ and CD34+ cell populations has not been performed in any species. Methods Immunohistochemistry was used to identify adventitial cells (CD34+) and pericytes (CD146+) and to determine their localization in relation to MSCs in equine tissues. Isolation of CD34+ (CD34+/CD146–/CD144–/CD45–) and CD146+ (CD146+/CD34–/CD144–/CD45–) cell fractions from equine adipose tissue was achieved by fluorescence-activated cell sorting. The isolated cell fractions were cultured and analyzed for the expression of MSC markers, using qPCR and flow cytometry, and for the ability to undergo trilineage differentiation. Angiogenic properties were analyzed in vivo using a chorioallantoic membrane (CAM) assay. Results Both CD34+ and CD146+ cells displayed typical MSC features, namely growth in uncoated tissue culture dishes, clonal growth when seeded at low density, expression of typical MSC markers, and multipotency shown by the capacity for trilineage differentiation. Of note, CD146+ cells were distinctly angiogenic compared with CD34+ and non-sorted cells (conventional MSCs), demonstrated by the induction of blood vessels in a CAM assay, expression of elevated levels of VEGFA and ANGPT1, and association with vascular networks in cocultures with endothelial cells, indicating that CD146+ cells maintain a pericyte phenotype in culture. Conclusion This study reports for the first time the successful isolation and culture of CD146+ and CD34+ cell populations from equine tissues. Characterization of these cells evidenced their distinct properties and MSC-like phenotype, and identified CD146+ cells as distinctly angiogenic, which may provide a novel source for enhanced regenerative therapies

The roles of immune cells in bone healing; what we know, do not know and future perspectives

Key events occurring during the bone healing include well-orchestrated and complex interactions between immune cells, multipotential stromal cells (MSCs), osteoblasts and osteoclasts. Through three overlapping phases of this physiological process, innate and adaptive immune cells, cytokines and chemokines have a significant role to play. The aim of the escalating immune response is to achieve an osseous healing in the shortest time and with the least complications facilitating the restoration of function. The uninterrupted progression of these biological events in conjunction with a favourable mechanical environment (stable fracture fixation) remains the hallmark of successful fracture healing. When failure occurs, either the biological environment or the mechanical one could have been disrupted. Not infrequently both may be compromised. Consequently, regenerative treatments involving the use of bone autograft, allograft or synthetic matrices supplemented with MSCs are increasingly used. A better understanding of the bone biology and osteoimmunology can help to improve these evolving cell-therapy based strategies. Herein, an up to date status of the role of immune cells during the different phases of bone healing is presented. Additionally, the known and yet to know events about immune cell interactions with MSCs and osteoblasts and osteoclasts and the therapeutic implications are being discussed

Adipose derived pericytes rescue fractures from a failure of healing – non-union

Atrophic non-union is attributed to biological failure of the fracture repair process. It occurs in up to 10% of fractures, results in significant morbidity to patients, and treatment often requires complex reconstructive procedures. We tested the ability of human bone derived marrow mesenchymal stem cells (MSC), and human adipose derived pericytes (the native ancestor of the MSC) delivered percutaneously to the fracture gap to prevent the formation of atrophic non-union in a rat model. At eight weeks, 80% of animals in the cell treatment groups showed evidence of bone healing compared to only 14% of those in the control group. Radiographic parameters showed significant improvement over the eight-week period in the cell treatment groups, and histology confirmed bone bridges at the fracture gap in the both treatment groups. The quality of bone produced and its biomechanical properties were significantly enhanced in both treatment groups. The results from this study demonstrate that MSC and pericytes have significant bone regeneration potential in an atrophic non-union model. These cells may have a role in the prevention of atrophic non-union and could enable a paradigm shift in the treatment of fractures at high risk of failing to heal and developing non-union

Isolation and characterization of canine perivascular stem/stromal cells for bone tissue engineering

For over 15 years, human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC). The isolation of perivascular progenitor cells from human adipose tissue by a cell sorting strategy was first published in 2008. Since this time, the interest in using pericytes and related perivascular stem/stromal cell (PSC) populations for tissue engineering has significantly increased. Here, we describe a set of experiments identifying, isolating and characterizing PSC from canine tissue (N = 12 canine adipose tissue samples). Results showed that the same antibodies used for human PSC identification and isolation are cross-reactive with canine tissue (CD45, CD146, CD34). Like their human correlate, canine PSC demonstrate characteristics of MSC including cell surface marker expression, colony forming unit-fibroblast (CFU-F) inclusion, and osteogenic differentiation potential. As well, canine PSC respond to osteoinductive signals in a similar fashion as do human PSC, such as the secreted differentiation factor NEL-Like Molecule-1 (NELL-1). Nevertheless, important differences exist between human and canine PSC, including differences in baseline osteogenic potential. In summary, canine PSC represent a multipotent mesenchymogenic cell source for future translational efforts in tissue engineering