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

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 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

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

Evaluating the Clinical Relevance of Codon 594 (G>A) Polymorphism of Estrogen Receptor Alpha in Knee Osteoarthritis

It has been reported that oestrogen receptor alpha (ER-?) polymorphisms are associated with knee osteoarthritis (OA). In this study, we assessed whether there was any association between the codon 594 (G>A) polymorphism in ER-? and radiographic features of OA or patient function.Radiographs, WOMAC score and patient reported time of symptom onset were assessed in 194 patients presenting for total knee replacement at Ramathibodi hospital over a one year period. ESR-1 genotyping was assessed. There were 107 (55.15%) patients with common homozygote (GG), 78 (40.20%) patients with heterozygote (GA) and nine (4.65%) patients with rare homozygote (AA). There was poor correlation (r = <0.2) between group difference in the radiographic parameters, time of onset of symptom , or in WOMAC scores. This polymorphism is not associated with the clinical features of knee osteoarthritis. The role of this polymorphism is unlikely then to be used as a biological marker predicting the progression of knee OA