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

Reconstruction of 10mm segmental bone defects in rat by mesenchymal stem cell derived chondrogenic cells (MSC-DC). Background. Mesenchymal stem cell derived condrogenic cells (MSC-DC) have excellent potential for healing 5 mm bone defect in rat femur. Purpose. To evaluate the effectiveness of MSC-DC on bone healing in 10 mm segmental bone defects in rat femur. Methods. 10 millimeter bone defects were produced in rat femur and fixed with external fixator. We divided this model into four groups according to the kind of graft for bone defects. These bone defects were grafted by MSC-DC seeded on a poly (DL-lactic acid-co-glycolic acid) (PLGA) scaffold in Group A, MSC seeded on a PLGA scaffold in Group B, PLGA scaffold only in Group C, and autologus bone graft in Group D. The healing processes were monitored radiographically and studied biomechanically and histologically. Results. All the bone defects in Group A healed radiographically with bridging callus formation at 4 weeks after the procedure, while none of Group B, C, and D had achieved bone union even at 8 weeks. Mechanical testing revealed that Group A showed approximately 40 % bending strength at 4 weeks compared with the contralateral side, and approximately 60 % at 8 weeks. In histology, Group A, maturation of bridging callus occurred from outside and enchondral ossification was prominent from inside. Conclusion. This study showed that MSC-DC with PLGA scaffold enhances bone healing even in large bone defects

Purpose. The purpose of this study was to evaluate the effects of implantation of mesenchymal stem cell derived condrogenic cells (MSC-DC) on bone healing in segmental defects in rat femur. Methods. Five-millimeter segmental bone defects were produced in the mid-shaft of the femur of Fisher 344 rats and stabilized with external fixator. The Treatment Group received MSC-DC, seeded on a PLGA scaffold, locally at the site of the bone defect, and Control Group received scaffold only. The healing processes were monitored radiographically (Softex), and studied radiographically (Micro-CT) and histologically. Results. All the bone defects in the Treatment Group healed radiographically with bridging callus formation at 4 weeks after the procedure, while none of the Control Group had achieved bone union. Micro-CT showed that newly formed bone volume in the Treatment Group at 16 weeks was 1.5 times that of unaffected side. Histological examination showed that the implanted scaffold of the Treatment Group were covered with periosteum-derived bridging callus and filled with cancellous bone-like tissue derived from enchondral ossification. Conclusion. The results of this study suggest that implantation of MSC-DC surprisingly enhances bone healing in segmental bone defects in rat much better than previously reported similar therapy using MSC

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

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

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

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.

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

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

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

Our unpublished data has indicated that the perivascular stem cells (PSCs) have increased chondrogenic potential compared to mesenchymal stem cells (MSCs) derived in culture. There has been a recent change in the theory that stem cells work by a paracrine effect rather than differentiation. There are minimal data demonstrating the persistence of implanted stem cells when used for engraftment. This study aimed to develop an autologous large animal model for perivascular stem cells as well as to determine if cells were retained in the articular cartilage defects. The reactivity of anti-human and anti-ovine antibodies was ascertained using immunohistochemistry and fluorescence-activated cell sorting (FACS). A panel of antibodies were combined and used to identify and purify pericytes (CD34-CD45-CD146+) and adventitial cells (CD34+CD45-CD146-) using FACS. The purified cells were cultured and their identity checked using FACS. These cultured cells demonstrated osteogenic, adipogenic and chondrogenic potential. Autologous ovine PSCs (oPSCs) were isolated, cultured and transfected using a GFP virus. The transfection rate was 88%. The cells were implanted into an articular cartilage defect on the medial femoral condyle using a hydrogel, four weeks following implantation the condyle was explanted and confocal laser scanning microscopy demonstrated the presence of oPSCs in the defect. Histology did not demonstrate any repair tissue at this early time point. These data have confirmed the viability our large animal model and that the implanted stem cells were retained in the defect four weeks following implantation

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

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

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. 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

Nanoscale topography increases the bioactivity of a material and stimulates specific responses (third generation biomaterial properties) at the molecular level upon first generation (bioinert) or second generation (bioresorbable or bioactive) biomaterials. We developed a technique (based upon the effects of nanoscale topography) that facilitated the in vitro expansion of bone graft for subsequent implantation and investigated the optimal conditions for growing new mineralised bone in vitro. Two topographies (nanopits and nanoislands) were embossed into the bioresorbable polymer Polycaprolactone (PCL). Three dimensional cell culture was performed using double-sided embossing of substrates, seeding of both sides, and vertical positioning of substrates. The effect of Hydroxyapatite, and chemical stimulation were also examined. Human bone marrow was harvested from hip arthroplasty patients, the mesenchymal stem cells culture expanded and used for cellular analysis of substrate bioactivity. The cell line specificity and osteogenic behaviour was demonstrated through immunohistochemistry, confirmed by real-time PCR and quantitative PCR. Mineralisation was demonstrated using alizarin red staining. Results showed that the osteoinduction was optimally conferred by the presence of nanotopography, and also by the incorporation of hydroxyapatite (HA) into the PCL. The nanopit topography and HA were both superior to the use of BMP2 in the production of mineralised bone tissue. The protocol from shim production to bone marrow harvesting and vertical cell culture on nanoembossed HaPCL has been shown to be reproducible and potentially applicable to economical larger scale production

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

Introduction. Nonunions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing, although the effects of different clinical ES waveforms available in clinical practice on BMMSCs cellular activities is unknown. Materials and Methods. We compared Direct Current (DC), Capacitive Coupling (CC), Pulsed Electromagnetic wave (PEMF) and Degenerate Wave (DW) by stimulating human-BMMSCs for 5 days for 3 hours a day. Cytotoxicity, cell proliferation, cell-kinetics and cell apoptosis were evaluated after ES. Migration and invasion were assessed using fluorescence microscopy and affected gene and protein expression were quantified. Results. DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms and unstimulated cells after 5 days of ES (p < 0.001). DC, DW and CC resulted in significantly more cells in S phase and G2/M phase (p < 0.01) compared to the unstimulated BMMSCs. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression (p < 0.001) compared to the other waveforms and unstimulated BMMSCs. DC increased cellular migration in a scratch-wound assay and all ES waveforms increased migration gene expression with DC having the greatest effect (p < 0.01). Conclusion. The ES waveform is vital in influencing BMMSCs cellular activities. Migration and invasion were increased by ES which suggests that the recruitment of BMMSCs to the healing site during a fracture could be increased by ES

Engineered bone tissue to recreate the continuity of damaged skeletal segments is one of the field of interest of tissue engineering. Trabecular titanium has very good mechanical properties and high in vitro and in vivo biocompatibility: it can be used in biomedical applications to promote osteointegration demonstrating that it can be successfully used for regenerative medicine in orthopaedic surgery (1). Purpose of this investigation was to evaluate the behavior of adipose tissue derived stem cells (hASCs) cultured on scaffolds of Trabecular TitaniumTM (Lima-Lto) (TT). hASCs are considered to be multipotent mesenchymal stem cells that are easily induced to differentiate into functional osteoblasts both in vitro and in vivo (2). The hASCs were obtained from the subcutaneous adipose tissue of healthy donors during total hip replacement procedures after digestion with collagenase. They were seeded on monolayer and on the TT scaffolds, and incubated at 37 degrees C in 5% CO2 with osteogenic medium or control medium. The expression of bone-related genes using RT-PCR, time course of alkaline phosphatase activity and morphological investigation with Scanning Electron Microscopy (SEM) were performed to evaluate the osteogenic differentiation of hASCs. Alkaline phosphatase activity, marker of the differentiation toward the osteogenic pattern, was significantly higher in hASCs grown with osteogenic medium than in cells grown with control medium, both in monolayer and TT scaffolds; moreover, also alkaline phosphatase of hASCs grown on TT scaffolds in the presence of control medium increased with time, differently from that of cells grown on monolayer. The osteogenic differentiated hASCs expressed the bone-related genes type I collagen, osteocalcin, Runx-2 and alkaline phosphatase. SEM observations showed that hASCs differentiated toward osteoblast-like cells: they produced a big amount of extracellular matrix that covered the surface of the porous scaffolds with bridges between the pore walls. These data suggest that hASCs are able to adhere to TT scaffolds, to acquire an osteoblastic phenotype and to produce abundant extracellular matrix, with but also without osteogenic medium. We can therefore conclude that this material carries osteinductive properties being responsible of ostegenic differentiation; consequently, this scaffold/cells construct is effective to regenerate damaged tissue and to restore the function of bone tissue

Introduction. Regenerative medicine is a rapidly expanding discipline. However due to a lack of validated outcome measures, clinical trials have been far few. This study aims to assess the validity, inter-observer reliability and intra-observer reproducibility of experimental fracture healing assessment on plain radiographies. This technique involves implantation of mesenchymal stem cell (MSC) seeded constructs on only one side of the fracture after randomisation. Methods. We examined inter/intraobserver agreement on the area and “bridging length” of callus formed on opposite sides of the fracture. Among 16 orthopaedic surgeons with trauma commitments (8 consultants, 8 registrars) on two separate occasions (average 52 days apart). They independently assessed the radiographs (AP or lateral) of 28 patients with fractures of the tibial or femoral shaft. The fractures chosen included non-unions treated with MSC/constructs and fresh fractures at 4–9 months. For each radiograph the assessor assigned which side (medial or lateral) is there more callus. Chase-corrected agreement using Fleiss kappa was used to compare opinions. Digital analysis software (Image-J) was used to quantify extent/bridging callus and correlate it with surgeons opinion. Results. Inter-observer variation showed a substantial overall agreement (k = 0.716) on the fracture side containing a larger “area” of callus but moderate agreement (k = 0.489) on side with more “bridging length”. These results were reproducible with a substantial overall intraobserver agreement. MSC/construct treated non-union showed a larger amount of agreement than fresh fractures for area (k = 0.754 vs 0.613) and bridging (0.550 vs 0.406). Utilizing digital analysis, non-unions showed a significant larger quantifiable difference between sides than fresh fractures (p = 0.009) for area but not bridging length (p = 0.269). Digital analysis quantification and surgeons opinion showed an almost perfect agreement for area (k = 0.867) and bridging (k = 0.846). Discussion. In this study we aimed to validate a novel method at studying the efficacy and effect of regenerative techniques on fracture healing. In particular, plain radiographs for comparing a treatment/internal control side. In this study we showed this method assessing area of callus is valid, reliable and reproducible. This is particularly so for MSC/construct treated non-union where the difference in both sides is higher as quantified in digital analysis. This is a novel method of experimental fracture healing using an internal control which decreases the variation between groups and sample size needed. This makes regenerative medicine clinical trials easier