Objectives. Nonunion is one of the most troublesome complications to treat in orthopaedics. Former authors believed that atrophic nonunion occurred as a result of lack of mesenchymal stem cells (MSCs). We evaluated the number and viability of MSCs in site of atrophic nonunion compared with those in iliac crest. Methods. We enrolled five patients with neglected atrophic nonunions of long bones confirmed by clinical examinations and plain radiographs into this study. As much as 10 ml bone marrow aspirate was obtained from both the nonunion site and the iliac crest and cultured for three weeks. Cell numbers were counted using a haemocytometer and vitality of the cells was determined by trypan blue staining. The cells were confirmed as MSCs by evaluating their expression marker (CD 105, CD 73, HLA-DR, CD 34, CD 45, CD 14, and CD 19). Cells number and viability were compared between the nonunion and iliac creat sites. Results. After three weeks, numbers of 6.08×10. 6. cells (. sd. 2.07) and 4.98×10. 6. cells (. sd. 1.15) were obtained from the nonunion site and the iliac crest, respectively, with viability of 87.1% (81.7% to 90.8%) and 89.8% (84.7% to 94.5%), respectively. No differences was found between the two sources of MSCs regarding cells number (p = 0.347) and viability (p = 0.175). Conclusions. Our findings showed the existence of MSCs in the site of atrophic nonunion, at a similar number and viability to those isolated from the iliac crest

The scarcity of mesenchymal stem cells (MSCs) in iliac crest bone marrow aspirate (ICBMA), and the expense and time in culturing cells, has led to the search for alternative harvest sites. The reamer-irrigation-aspirator (RIA) provides continuous irrigation and suction during reaming of long bones. The aspirated contents pass via a filter, trapping bony fragments, before moving into a ‘waste’ bag from which MSCs have been previously isolated. We examined the liquid and solid phases, performed a novel digestion of the solid phase, and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA. The solid fraction from the filtrate was digested for 60 minutes at 37°C with collagenase. Enumeration was performed via the colony-forming unit fibroblast (CFU-F) assay. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages, and their phenotypes assessed using flow cytometry (CD33, CD34, CD45, CD73, CD90, and CD105). MSCs from the RIA phases were able to differentiate at least as well as those from ICBMA, and all fractions had phenotypes consistent with other established sources. The median number of colonies for the three groups was: ICBMA = 8.5 (2 to 86), RIA-liquid = 19.5 (4 to 90), RIA-solid = 109 (67 to 200) per 200 μl. The mean total yield of cells for the three groups was: ICBMA = 920 (0 to 4275), RIA-liquid = 114 983 (16 500 to 477 750), RIA-solid = 12 785 (7210 to 28 475). The RIA filtrate contains large numbers of MSCs that could potentially be extracted without enzymatic digestion and used for bone repair without prior cell expansion

As arthroplasty demand grows worldwide, the need for a novel cost-effective treatment option for articular cartilage (AC) defects tailored to individual patients has never been greater. 3D bioprinting can deposit patient cells and other biomaterials in user-defined patterns to build tissue constructs from the “bottom-up,” potentially offering a new treatment for AC defects. Novel composite bioinks were created by mixing different ratios of methacrylated alginate (AlgMA) with methacrylated gelatin (GelMA) and collagen. Chondrocytes and mesenchymal stem cells (MSCs) were then encapsulated in the bioinks and 3D bioprinted using a custom-built extrusion bioprinter. UV and double-ionic (BaCl2 and CaCl2) crosslinking was deployed following bioprinting to strengthen bioink stability in culture. Chondrocyte and MSC spheroids were also bioprinted to accelerate cell growth and development of ECM in bioprinted constructs. Excellent viability of chondrocytes and MSCs was seen following bioprinting (>95%) and maintained in culture, with accelerated cell growth seen with inclusion of cell spheroids in bioinks (p<0.05). Bioprinted 10mm diameter constructs maintained shape in culture over 28 days, whilst construct degradation rates and mechanical properties were improved with addition of AlgMA (p<0.05). Composite bioinks were also injected into in vitro osteochondral defects and crosslinked in situ, with maintained cell viability and repair of osteochondral defects seen over a 14-day period. In conclusion, we developed novel composite bioinks that can be triple-crosslinked, facilitating successful chondrocyte and MSC growth in 3D bioprinted scaffolds and in vitro repair of an osteochondral defect model. This offers hope for a new approach to treating AC defects

Mesenchymal stem cells (MSCs) reside around blood vessels in all organs. This reservoir of progenitors can be ‘recruited’ in response to injury. The ability to manipulate stem cells therapeutically within injured tissue provides an attractive alternative to transplantation. Stem cells are regulated by neighbouring cells. We hypothesized that endothelial cells (ECs) influence MSC differentiation into bone and fat. MSCs were sorted from fat using fluorescent activated sorting. Their capacity to differentiate into bone, fat and cartilage was used to confirm MSC phenotype. MSCs and ECs were cultured in two-dimensions (standard culture dishes) and three-dimensions (vascular networks suspended in gel). Cocultures were exposed to osteogenic and adipogenic media. The role of EC-released factors on MSC differentiation was determined using a system in which cells share media but do not contact. Wnt pathway modulators were used to investigate the role of Wnt signalling. MSCs differentiated into bone, fat and cartilage. MSCs and ECs integrated in two- and three-dimensions. MSCs and ECs formed vessel-like structures in three-dimensions. When cultured with ECs, MSC differentiation to bone was accelerated while differentiation to fat was inhibited. This effect on osteogenesis was maintained when cells shared media but did not contact. Coculture with Wnt modulators confirmed that this effect is in part, mediated through Wnt signalling. Our data suggest that ECs influence MSC differentiation. Therapeutic targeting of EC-MSCs signalling may enable manipulation of MSCs in vivo avoiding the need for cell transplantation. This could enable trauma and orthopaedic patients who have healthy resident stem cells to self-repair

Introduction. MSCs have long promised benefits of synthesising bone/cartilage, treating non-unions and potentially accelerating fracture repair. This potential has been tempered by MSC scarcity in the ‘gold-standard’ iliac crest bone marrow aspirate (ICBMA) and the resulting need to expand numbers via cell-culture. Culture of MSCs is time-consuming, expensive and results in cells with a reduced differentiation capacity. The reamer-irrigator-aspirator (RIA) is an innovation designed to reduce intra-medullary (IM) pressures during reaming of long-bones via continuous irrigation and suction. Aspirated contents are passed via a coarse filter, which traps bony-fragments before moving into a ‘waste’ bag - from which MSCs have been previously isolated. We examined liquid and solid phases found in this ‘waste’, performed a novel digestion of the solid phase and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA. Methods. The filtrate ‘waste’ bag from RIA reaming (6 patients) was filtered (70μm) and the solid fraction digested for 60min (37°C) with collagenase. MSCs were isolated from liquid & solid fractions and from 10ml matched ICBMA. Enumeration of MSCs was achieved via colony-forming-unit-fibroblast (CFUF) assay and flow-cytometry on fresh sample using CD45low, CD271+. MSCs were cultured by virtue of their plastic adherence and passaged in standard, non-haematopoietic media. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages with their phenotype assessed with flow cytometry CD33 CD34 CD45 CD73 CD90 CD105. Results. We found MSCs were in all fractions/patients. Using the CFU-F assay median number of colonies: ICBMA=8 (2–21), RIA-liquid=12 (4–41), RIA-solid=115 (67–200) per 200μl of sample. Total yield of cells was calculated from volume of sample: ICBMA=670 (228–4275), RIA-liquid=39000 (16500–83700), RIA-solid=9400 (7210–28475). MSC frequency as a percentage of total cells using flow-cytometry on fresh sample found similar frequencies. MSCs isolated from the RIA phases differentiated into osteogenic, chondrogenic and adipogenic lineages at least as well as ICBMA. Passaged (P2) cells, from all fractions/patients, had a phenotype consistent with other reported sources. Discussion. The RIA filtrate bag is typically discarded at operation. These results show that this ‘waste’ represents a significant source of MSCs that could be isolated for autologous/allogenous use. Concentration of the liquid-phase/brief enzymatic digestion of the solid-phase offers the possibility of large numbers of MSCs being obtained without/with minimal culture expansion

The role of mesenchymal stem cells (MSCs) in enhancing healing process has been examined with allogeneic and xenogeneic cells in transplantation models. However, certain factors might limit the use of allogeneic cells in clinical practice, (e.g. disease transmission, ethical issues and patient acceptance). Adipose tissue represents an abundant source for autologous cells. The aim of this study was to evaluate adipose-derived autologous cells for preventing non-union. Adults male Wistar rats (n=5) underwent a previously published surgical procedure known to result in non-union if no treatment is given. This consisted of a mid-shaft tibial osteotomy with peri/endosteal stripping stabilised by intramedullary nail fixation with a 1mm gap maintained by a spacer. During the same operation, ipsilateral inguinal subcutaneous fat was harvested and processed for cell isolation. After three weeks in culture, the cell number reached 5×106 and were injected into the fracture site. At the end of the experiment, all tibias (injected with autologous fat-MSCs) developed union. These were compared with a control group injected with PBS (n=4) and with allogenic (n=5) and xenogeneic (n=6) cell transplantation groups. The amount of callus was noticeably large in the autologous cell group and the distal-callus index was significantly greater than that of the other groups, P-value =<0.05, unpaired t-test, corrected by Benjamini & Hochberg. We report a novel method for autologous MSCs implantation to stimulate fracture healing. Local injection of autologous fat-MSCs into the fracture site resulted in a solid union in all the tibias with statistically significantly greater amounts of callus

Bone is the second most commonly transplanted tissue worldwide, with over four million operations using bone grafts or bone substitute materials annually to treat bone defects. However, significant limitations affect current treatment options and clinical demand for bone grafts continues to rise due to conditions such as trauma, cancer, infection and arthritis. The need for a novel, cost effective treatment option for osteochondral defects has therefore never been greater. As an emerging technology, three-dimensional (3D) bioprinting has the capacity to deposit cells, extracellular matrices and other biological materials in user-defined patterns to build complex tissue constructs from the “bottom up”. Through use of extrusion bioprinting and fused deposition modelling (FDM) 3D printing, porous 3D scaffolds were successfully created in this study from hydrogels and synthetic polymers. Mesenchymal stem cells (MSCs) seeded onto polycaprolactone scaffolds with defined pore sizes and porosity maintained viability over a 7-day period, with addition of alginate hydrogel and scaffold surface treatment with NaOH increasing cell adhesion and viability. MSC-laden alginate constructs produced via extrusion bioprinting also maintained structural integrity and cell viability over 7 days in vitro culture. Growth within osteogenic media resulted in successful osteogenic differentiation of MSCs within scaffolds compared to controls (p<0.001). MSC spheroids were also successfully created and bioprinted within a novel, supramolecular hydrogel with tunable stiffness. In conclusion, 3D constructs capable of supporting osteogenic differentiation of MSCs were biofabricated via FDM and extrusion bioprinting. Future work will look to increase osteochondral construct size and complexity, whilst maintaining cell viability

Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) as it is largely dispensable and readily accessible through minimally invasive procedures such as lipoaspiration. Until recently MSCs could only be isolated in a process involving ex-vivo culture. Pericytes (CD45−, CD146+, and CD34−) and adventitial cells (CD45−, CD146−, CD34+) represent two populations of MSCs (collectively termed perivascular stem cells or PSCs) that can be prospectively purified using fluorescence activated cell sorting (FACS). We performed FACS on lipoaspirate samples from n=129 donors to determine the frequency and yield of PSCs and to establish patient and processing factors that influence yield. The mean number of stromal vascular fraction (SVF) cells from 100ml of lipoaspirate was 37.8×106. Within the SVF, mean cell viability was 82%, with 31.6% of cells being heamatopoietic (CD45+). Adventitial cells and pericytes represented 31.6% and 7.9% of SVF cells respectively. As such, 200ml of lipoaspirate would theoretically yield 24.5 million MSCs –a sufficient number to enable point-of-care delivery for use in several orthopaedic applications. The yield and prevalence of PSCs were minimally affected by donor age, sex and BMI. Storing lipoaspirate samples for up to 72 hours prior to processing had no significant deleterious effects on MSC yield or viability. Our study confirms that pure populations of MSC-precursors (PSCs) can be prospectively isolated from adipose tissue, in sufficient quantities to negate the necessity for culture expansion while widening possible applications to include trauma, where a time delay between extraction and implantation excludes their use

Perivascular stem cells (PSCs) from lipoaspirate demonstrate increased purity and immaturity with greater engraftment potential than standard mesenchymal stem cells (MSCs). MSCs from the infra-patellar fat pad (IFP) have previously demonstrated increased chondrogenic potential. This study investigated the availability and potential of PSCs harvested from the infra-patellar fat pad of the human knee for musculoskeletal regeneration. Tissue sections of IFP were stained with markers for PSCs, MSCs and endothelial cells to confirm their presence and location. Samples were obtained from patients undergoing TKR (n=13) or ACL reconstructions (n=10). Pericytes and adventitial cells made up 3.8% and 21.2% respectively of the stromal vascular fraction. The total number of pericytes and adventitial cells were 4.6±2.2×104 and 16.2±3.2×104 respectively. Cells were cultured both separately and combined. Cell identity was ascertained using fluorescence-activated cell sorting, immunocytochemistry and PCR. Cultured PSCs were differentiated using chondrogneic, osteogenic, adipogenic and myogenic medias. Differentiation was determined using Alcian Blue, Alizarin red, Oil Red O and myosin staining. This study demonstrates that the IPFP is a viable source of PSCs that can be harvested either arthroscopically or through an arthrotomy by orthopaedic surgeons for cell-based musculoskeletal regeneration. Their potential now needs to be compared to conventional MSCs

Perivascular stem cells (PSCs) from lipoaspirate demonstrate increased purity and immaturity with greater engraftment potential than standard mesenchymal stem cells (MSCs). MSCs from the infra-patellar fat pad (IFP) have previously demonstrated increased chondrogenic potential. This study investigated the availability and potential of PSCs harvested from the infra-patellar fat pad of the human knee for musculoskeletal regeneration. Sections of IFP were stained with markers for PSCs, MSCs and endothelial cells to confirm their presence and location. Samples were obtained from patients undergoing TKR (n=13) or ACL reconstructions (n=10). Pericytes and adventitial cells made up 3.8% and 21.2% respectively of the stromal vascular fraction. The total number of pericytes and adventitial cells were 4.6±2.2×10. 4. and 16.2±3.2×10. 4. respectively. Cells were cultured both separately and combined. Cell identity was ascertained using fluorescence-activated cell sorting and immunocytochemistry. Cultured PSCs were differentiated using chondrogneic, osteogenic, adipogenic and myogenic medias. Differentiation was determined using Alcian Blue, Alizarin red, Oil Red O and mysosin staining. This study demonstrates that the IFP is a viable source of PSCs that can be harvested either arthroscopically or through an arthrotomy by orthopaedic surgeons for cell-based musculoskeletal regeneration. Their potential now needs to be compared to conventional MSCs

Introduction. The concept of “bone graft expanders” has been popularised to increase the volume and biological activity of the implanted Material. HYPOTHESIS. Orthoss® granules support exogenously seeded MSCs and attract neighbouring host MSCs. Methods. In 3-D cultures’ Orthoss® granules were seeded with 2×10. 5. bone marrow MSCs/granule and maintained in MSC expansion or differentiation media for 21 days. In homing experiments’ bone autografts were placed in close proximity to Orthoss®. Scaffold colonisation and MSC differentiation were assessed by confocal microscopy’ standard electron microscopy’ and energy-dispersive X-ray spectroscopy. Results. Long-term incubation of MSC/scaffold resulted in formation of multiple cell-matrix layers lining the scaffold pores as well as outer surfaces. MSC differentiation to osteoblasts was evident as strong deposition of Calcium and Phosphorus was detected in both MSC expansion and osteogenic conditions. Cell egress experiments demonstrated the migration of cells from neighbouring autografts and their attachment and re-settlement on Orthoss®. Discussion & Conclusions. Orthoss® scaffolds support MSC attachment’ growth and osteogenic differentiation whereas resident bone subpopulations can rapidly migrate towards’ attach’ and expand on them. These results indicate that Orthoss® can serve as a graft expander for repairing large bone defects in trauma patients

Introduction. Iliac crest bone marrow aspirate (ICBMA) is frequently cited as the ‘gold-standard’ source of MSCs. Mesenchymal stem cells have been shown to reside within the intramedullary (IM) cavities of long-bones and a comparative assessment with ICBMA has not yet been performed. Methods. Aspiration of the IM cavities of 6 patients' femurs with matched ICBMA was performed. The long-bone-fatty-bone-marrow (LBFBM) aspirated was filtered (70μm) and the solid fraction digested for 60min (37°C) with collagenase. Enumeration was performed via the colony-forming-unit-fibroblast (CFU-F) assay and using the CD45low CD271+ phenotype via flow-cytometry. Passaged (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages with their phenotype assessed using flow-cytometry CD33 CD34 CD45 CD73 CD90 CD105. Results. MSCs were isolated from all fractions. Using the CFU-F assay median number of colonies: ICBMA=8 (2–21), LBFBM-liquid=14 (0–53), LBFBM-solid=116 (23–171) per 200μl of sample; MSC frequency, as percentage of total cells, using flow-cytometry, provided similar results. Mesenchymal stem cells isolated from the LBFBM phases appeared to not be inferior to ICBMA in terms of osteogenic, chondrogenic or adipogenic differentiation. Passaged cells from all fractions had a phenotype consistent with other reported sources. Discussion. Intra-medullary cavities of long-bones are frequently accessed by the orthopaedic/trauma surgeon. This represents a ‘low-tech’ method of harvesting large numbers of MSCs with a favourable differentiation profile for autologous/allogenous uses

We hypothesise that the Masquelet induced membrane used for the reconstruction of large bone defects were likely to involve mesenchymal stem cells (MSCs), given the excellent resultant skeletal repair. This study represents the first characterisation in humans of the induced membrane formed as a result of the Masquelet technique. Methods. Induced membranes and matching periosteum were harvested from 7 patients. Cytokines (BMP2, VEGF, SDF1) and cell lineage markers (CD31, CD271, CD146) were studied by immunohistochemisty. Flow cytometry was used to measure the cellularity and cellular composition. MSCs were enumerated using a colony forming unit fibroblast assay. In expanded cultures, a 96-gene array card was used to assess their transcriptional profile. Alkaline phophatase, alizarin red and calcium assays were employed to measure their in vitro osteogenic potential. Results. Membrane was more cellular(p=0.028), had more MSC phenotype(p=0.043) compared to matched periosteum. The molecular profiles were similar, except for 2-fold abundance of SDF-1 in membrane (p=0.043)compared to periosteum. Membrane and periosteum had a similar proportion of endothelial cells and CFU-F colonies; expanded MSCs from both sources were highly osteogenic. Discussion. These results indicate that the induced membrane possesses a rich source of MSC and therefore our findings support the view that the induced membrane plays an active role in bone regeneration

Proliferation of synovial Mesenchymal Stromal/Stem Cells (MSCs) leads to synovial hyperplasia (SH) following Joint Surface Injury (JSI). Uncontrolled Yap activity causes tissue overgrowth due to modulation of MSC proliferation. We hypothesised that YAP plays a role in SH following JSI. A spatiotemporal analysis of Yap expression was performed using the JSI model in C57Bl/6 mice. Synovial samples from patients were similarly analysed. Gdf5-Cre;Yap1fl/fl;Tom mice were created to determine the effect YAP1 knockout in Gdf5 lineage cells on SH after JSI. In patients, Yap expression was upregulated in activated synovium, including a subset of CD55 positive fibroblast-like synoviocytes in the synovial lining (SL). Cells staining positive for the proliferation marker Ki67 expressed active YAP. In mice, Yap was highly expressed in injured knee joint synovium compared to controls. Yap mRNA levels at 2 (p<0.05) and 8 days (p<0.001) after injury were increased. Conditional Yap1 knockout in Gdf5 progeny cells prevented hyperplasia of synovial lining (SL) after JSI. Cellularity was significantly decreased in the SL but not in the sub-lining of injured Yap1 knockout- compared to control mice. The percentage of cells in synovium that were Tom+ increased in response to JSI in control and haplo-insufficient but not in YAP1 knockout mice (p<0.05). Modulation of YAP and proliferation of MSCs in the synovium after JSI provides a system to study the role of SH after trauma in re-establishing joint homeostasis and is a potential novel therapeutic target for the treatment of post traumatic OA

Exercise deters systemic diseases such as osteoporosis, sarcopenia, diabetes and obesity. Brief daily periods of low intensity vibration (LIV; <0.4g) is anabolic to bone and muscle, an adaptive response achieved in part by biasing mesenchymal stem cell (MSC) fate selection towards forming higher order connective tissues. In the clinic, LIV has protected the musculoskeletal system even under severe challenges such as Crohn Disease, Cerebral Palsy, and end-stage renal disease. Low magnitude mechanical signals also suppress adipogenesis in the mouse, with reductions in subcutaneous and visceral fat. The starkly distinct response of these tissues (augment bone & muscle; suppress fat) suggests that LIV influences the differentiation pathway of MSCs. Extending this diet induced obesity model to 7 months increased total adiposity, accelerated age-related loss of trabecular bone and severely reduced B & T-cell number in the marrow and blood, shifting hematopoietic stem cells (HSC) towards the myeloid lineage. LIV introduced at 4 months rescued bone and B-cells to those levels measured in regular diet controls. These data emphasise why inactivity can promote osteoporosis, diabetes and obesity, and why a sedentary individual is predisposed to disease sequelae. Protection of MSC and HSC populations by mechanical signals may represent a unique strategy by which adiposity can be suppressed, the immune system protected, and a musculoskeletal system enhanced

MicroRNAs (miRNAs ) are small non-coding RNAs that regulate gene expression. We hypothesised that the functions of certain miRNAs and changes to their patterns of expression may be crucial in the pathogenesis of nonunion. Healing fractures and atrophic nonunions produced by periosteal cauterisation were created in the femora of 94 rats, with 1:1 group allocation. At post-fracture days three, seven, ten, 14, 21 and 28, miRNAs were extracted from the newly generated tissue at the fracture site. Microarray and real-time polymerase chain reaction (PCR) analyses of day 14 samples revealed that five miRNAs, miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p, were highly upregulated in nonunion. Real-time PCR analysis further revealed that, in nonunion, the expression levels of all five of these miRNAs peaked on day 14 and declined thereafter.

Our results suggest that miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p may play an important role in the development of nonunion. These findings add to the understanding of the molecular mechanism for nonunion formation and may lead to the development of novel therapeutic strategies for its treatment.

Cite this article: Bone Joint J 2015; 97-B:1144–51.

We hypothesised that cells obtained via a Reamer–Irrigator–Aspirator (RIA) system retain substantial osteogenic potential and are at least equivalent to graft harvested from the iliac crest. Graft was harvested using the RIA in 25 patients (mean age 37.6 years (18 to 68)) and from the iliac crest in 21 patients (mean age 44.6 years (24 to 78)), after which ≥ 1 g of bony particulate graft material was processed from each. Initial cell viability was assessed using Trypan blue exclusion, and initial fluorescence-activated cell sorting (FACS) analysis for cell lineage was performed. After culturing the cells, repeat FACS analysis for cell lineage was performed and enzyme-linked immunosorbent assay (ELISA) for osteocalcin, and Alizarin red staining to determine osteogenic potential. Cells obtained via RIA or from the iliac crest were viable and matured into mesenchymal stem cells, as shown by staining for the specific mesenchymal antigens CD90 and CD105. For samples from both RIA and the iliac crest there was a statistically significant increase in bone production (both p < 0.001), as demonstrated by osteocalcin production after induction.

Medullary autograft cells harvested using RIA are viable and osteogenic. Cell viability and osteogenic potential were similar between bone grafts obtained from both the RIA system and the iliac crest.

Cite this article: Bone Joint J 2013;95-B:1269–74.