Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), a potential mechanism of MSC therapies has been attributed to paracrine activity, in which extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor compete with adult MSC in their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSC) appear to provide a promising source. In this study, MSCs and iMSC during long term-expansion using a serum free clinical grade condition, were characterized for surface expression pattern, proliferation and differentiation capacity, and senescence rate. Culture media were collected continuously during cell expansion, and EVs were isolated. Nanoparticle tracking analysis (NTA), transmission electron microscopy, western blots, and flow cytometry were used to identify EVs. We evaluated the biological effects of MSC and iMSC-derived EVs on human chondrocytes treated with IL-1α, to mimic the OA environment.

In both cell types, from early to late passages, the amount of EVs detected by NTA increased significantly, EVs collected during cells expansion, retained tetraspanins (CD9, CD63 and CD81) expression. The anti-inflammatory activity of MSC-EVs was evaluated in vitro using OA chondrocytes, the expression of IL-6, IL-8 and COX-2 was significantly reduced after the treatment with hMSC-derived EVs isolated at early passage. The miRNA content of EVs was also investigated, we identify miRNA that are involved in specific biological function.

At the same time, we defined the best culture conditions to maintain iMSC and define the best time window in which to isolate EVs with highest biological activity.

In conclusion, a clinical grade serum-free medium was found to be suitable for the isolation and expansion of MSCs and iMSC with increased EVs production for therapeutic applications.

Acknowledgments: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671

Bone marrow stem cells (BMSCs) represent a collection of different cell types exhibiting stem cell characteristics but with notable heterogeneity. Among these, Skeletal Stem Cells (SSCs) represent a distinct matrix subgroup within BMSC and demonstrate a specialized capacity to facilitate bone formation, recruit chondrocytes, and contribute to hematopoiesis. SSCs play a pivotal role in orchestrating the functions of skeletal organs. Local ischemia has a significant impact on cell survival and function. We hypothesize that bone ischemia induces alterations in the differentiation potential of SSCs, consequently influencing changes in bone structure. We mechanically dissected tissue from the necrotic segment in the femoral head and more normal appearing areas from the femoral neck of specimens from 5 patients diagnosed with osteonecrosis of the femoral head (ONFH). These tissues were enzymatically broken down into individual cell suspensions. Utilizing fluorescence-activated cell sorting (FACS) based on specific surface markers indicative of human skeletal stem cells (hSSC), namely CD45- CD235a- CD31- TIE2- Podoplanin (PDPN)+ CD146- CD73+ CD164+, we isolated a distinct cell population. Subsequent in vitro evaluations, focusing on clonogenicity, osteogenesis, and chondrogenesis were conducted to assess the functional prowess of these SSCs. Moreover, we introduced BMP2 at a concentration of 50ng/ml to SSCs extracted from necrotic regions to potentially reinstate their osteogenic capabilities. We effectively isolated SSCs from both Necrotic and Non-necrotic Zones. We observed an augmented clonal formation capacity and chondrogenesis ability of SSCs isolated from the necrotic region, accompanied by a significant decline in osteogenic ability (P＜0.01), an effect not reversible even with the addition of BMP2. Ischemia adversely affects the proliferation and function of SSCs, resulting in a diminished osteogenic capacity and an insensitivity to BMP2, ultimately leading to structural alterations in bone tissue

Introduction and Objective. Platelet-Rich-plasma (PRP) has been used in combination with stem cells, from different sources, with encouraging results both in vitro and in vivo in osteochondral defects management. Adipose-derived Stem Cells (ADSCs) represents an ideal resource for their ease of isolation, abundance, proliferation and differentiation properties into different cell lineages. Furthermore, Stem Cells in the adipose tissue are more numerous than from other sources. Aim of this study was to evaluate the potential of ADSCs in enhancing the effect of arthroscopic mesenchymal stimulation combined with infiltration of PRP. Materials and Methods. The study includes 82 patients. 41 patients were treated with knee arthroscopy, Steadman microfractures technique and intraoperative PRP infiltration, Group A. In the Group B, 41 patients were treated knee arthroscopy, Steadman microfractures and intraoperative infiltration of PRP and ADSCs (Group B). Group A was used as a control group. Inclusion criteria were: Age between 40 and 65 years, Outerbridge grade III-IV chondral lesions, Kellegren-Lawrence Grade I-II. Patient-reported outcome measures (PROMs) evaluated with KOOS, IKDC, VAS, SF-12 were assessed pre-operatively and at 3 weeks, 6 months, 1-year post-operative. 2 patients of Group A and 3 patients of Group B, with indication of Puddu plate removal after high tibial osteotomy (HTO), underwent an arthroscopic second look, after specific informed consent obtained. On this occasion, a bioptic sample was taken from the repair tissue of the chondral lesion previously treated with Steadman microfractures. Results. PROMs showed statistically significant improvement (p <0.05) with comparable results in both groups. The histological examination of the bioptic samples in Group B showed a repair tissue similar to hyaline cartilage, according to the International Cartilage Repair Society (ICRS) Visual Histological Assessment Scale. In Group A, the repair tissue was fibrocartilaginous. Conclusions. According to the PROMs and the histological results, showing repair tissue after Steadman microfractures qualitatively similar to hyaline cartilage, the combination of ADSCs and PRP could represent an excellent support to the arthroscopic treatment of focal chondral lesions and mild to moderate osteoarthritis

Introduction. Autologous Chondrocyte Implantation (ACI) is an effective surgical treatment for chondral defects. ACI involves arthrotomy for cell implantation. We describe the development of an intra-articular injection of cultured MSC, progressing from in-vitro analysis, through animal model, clinical and radiological outcome at five years follow up. Materials and Methods. We prospectively investigated sixteen patients with symptomatic ICRS grade III and IV lesions. These patients underwent cartilage repair using cultured mesenchymal stem cell injections and are followed up for five years. Results. Statistically significant clinical improvement was noted by two years and was sustained for five years of the study. At five years, mean Lysholm score was 80, compared to 44 pre-operatively. Symptomatic KOOS improved to 88 from 55. Subjective IKD Calso showed improvement from 42 to 76. On morphological MRI MOCART score was 76 and qualitative MRI showed the mean T2relaxation-times were 28 and 31 for their pair tissue and native cartilage respectively. Discussion. Cultured MSC provides a good number of uncommitted stem cells to the previously prepared chondral defects of the knee by “homing on” phenomenon. Cultured cells, suspended in serum can be delivered by an intra-articular injection. Conclusion. Use of cultured MSC is less invasive, avoids complications associated with arthrotomy, compared to ACI technique. Good clinical results were found to be sustained at five years of follow-up with a regenerate that appears like surrounding native cartilage. The use of Cultured Mesenchymal Stem Cells (MSC) has represented a promising treatment to restore the articular cartilage

Introduction There are 1 million cases of major skeletal defects :that occur worldwide each year that lead to significant morbidity and disability and currently require bone grafting as the main mode of treatment. Limitations of bone-grafting include donor site morbidity, reduced osseoinductivity and risk of pathogen transmission to the host. There is considerable interest in finding ways of differentiating mesenchymal stem cells down the osteoblastic lineage to form bone tissue. We hypothesized that there is an optimum strain that promotes differentiation of mesenchymal stem cells into osteoblasts. Methods: A bioreactor was developed that was capable of applying tensional forces across a culture strip in a graduated manner within a range of 1-4373me. Mesenchymal stem cells were grown on these strips and subjected to cyclical tensile strain at 1Hz. Cell morphology using Scanning Electron Microscopy, mineralization using specialized stains and expression of core binding factor1 (Cbfa1) was studied at various strain levels. Results: Scanning Electron Microscopy revealed classic osteoblastic cells in the regions subjected to tensile force, especially in the region where average strain was 1312me. X-ray microanalysis revealed calcium deposits on the strip, indicating osteoblastic differentiation. Cbfa1 expression was greatest in the region with an average strain 1312 me followed by a region on the strip subjected to just fluid shear without any tension. Cbfa1 expression was significantly greater in cells subjected to tensile forces than unstrained controls at all levels of strain tested (p< 0.05). Cbfa1 expression was further enhanced significantly by the addition of osteogenic factors (p< 0.05). Significantly greater mineralization (p< 0.05) occurred in the regions subject to tension with the greatest being in the region with an average strain of 1312 me. Conclusions: Mechanical tensile forces especially in the range of up to 2173me promote differentiation of Mesenchymal Stem Cells into osteoblasts and encourage expression of the Cbfa1 gene. Tensile strain also promotes mineralization. Chemical factors in form of osteogenic media accelerate the differentiation of MSCs and encourages earlier production of osteoblast specific markers. Fluid shear appears to have a beneficial effect in stimulating differentiation into the osteoblast phenotype and, combined with tensile strain, may offer an even greater osteogenic stimulus

The aim of the ongoing projects was to demonstrate the efficacy of autologous bone marrow derived stem cells (MSC) combined with biomaterial to induced new bone formation in a randomized multicenter controlled clinical trial.

Patients with a need for bone reconstruction of residual edentulous ridges in both the mandible and maxilla due to bone defects with a vertical loss of alveolar bone volume and/or knife edge ridges (≤ than 4,5 mm) unable to provide adequate primary stabilization for dental implants were included in the clinical study. Autologous bone marrow MSC were expanded, loaded on BCP and used to augment the alveolar ridges. After five months bone biopsies were harvested at the implant position site and implants were installed in the regenerated bone. The implants were loaded after 8–12 weeks. Safety, efficacy, quality of life and success/survival were assessed. Five clinical centers, 4 different countries participated. Bone grafts harvested from the ramus of the mandibles were used as control in the projects.

Summary. In this study, we challenged the current paradigm of human Mesenchymal Stem Cells survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to their survival. Introduction. The survival of human mesenchymal stem cells (hMSCs) has elicited a great deal of interest, because it is relevant to the efficacy of engineered tissues. However, to date, hMSCs have not met this promise, in part due to the high death rate of cells upon transplantation. In this study, we challenged the current paradigm of hMSC survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to hMSC survival. Materials and methods. In vitro model of ischemia 2.10. 4. hMSCs from five donors, were seeded into individual wells of a 24-well plate, cultured overnight, washed twice with PBS and then maintained in hypoxia (0.1% oxygen) under serum (FBS) free αMEM medium in either the absence or in the presence (1 or 5 g/L) of glucose for 21 days. In vitro Cell viability: To assess the role of glucose on hMSCs viability, cells were cultured under hypoxia in the absence or in the presence of glucose (1 and 5g/L), At days 0, 3, 7, 14 and 21, cell viability was evaluated by flow cytometry and ATP content per cell quantified. In vivo effect of glucose supply on hMSCs viability. 3.10. 5. eGFG-luc hMSCs were seeded on a cylindrical AN-69 scaffolds. At the time of implantation, 100 µl of hyaluronic acide (HA) (2%) containing either 0g/L (negative control) or 10g/L of glucose was gently injected inside the construct. Cell- constructs were implanted subcutaneously in eight week-old mice (2 per animal) and were imaged by bioluminescence imaging (BLI) at day 1, 4, 7 and 14 until sacrifice. Results. hMSCs were able to survive and to maintain their ATP content 21 days under sustained hypoxia providing that they were cultured in the presence of a sufficient glucose supply (i.e. 5g/L). In contrast, hMSCs cultured without or with 1g/L of glucose failed to survive. These results established that glucose depletion but not sustained hypoxia affected cell survival. In vivo results showed a striking increase of cell viability in cell constructs loaded with glucose. At day 14, a five-fold increase in cell number was observed in cell constructs loaded with glucose when compared to the control cell constructs without glucose. Discussion. The present study challenge the current paradigm that gives a pivotal role to oxygen on hMSCs massive cell death. By using an in vitro model of hypoxia/ischemia, we demonstrated that in the presence of sufficient glucose, hMSCs were able to survive 21 days under sustained hypoxia. Most importantly, an appropriate glucose supply strongly increases cell viability of hMSCs implanted subcutaneously in a mice model. This study provides evidences that glucose depletion but not hypoxia affects hMSCs viability. Further investigations need to be performed to develop hydrogels that ensure continuous glucose delivery to the implanted cells. Theses findings are particularly relevant because they pave the way to the development of new delivery systems to ensure hMSCs viability in order to increase their therapeutical potential after implantation

We found that adipose stem cells are poorly differentiated into bone and that their ability to differentiate into bone varies from cell line to cell line. The osteogenic differentiation ability of the adipose stem cell lines was distinguished through Alzarin Red Staining, and the cell lines that performed well and those that did not were subjected to RNA-seq analysis. The selected gene GSTT1 (glutathione S-transferase theta-1) gene is a member of a protein superfamily that catalyzes the conjugation of reduced glutathione to a variety of hydrophilic and hydrophobic compounds. The purpose of this study is to treat avascular necrosis and bone defect by improving bone regeneration with adipose stem cells introduced with a new GSTT1 gene related to osteogenic differentiation of adipose stem cells. In addition, the GSTT1 gene has the potential as a genetic marker that can select a specific cell line in the development of an adipose stem cell bone regeneration drug.

Total RNA was extracted from each sample using the TRIzol reagent. Its concentration and purity were determined based on A260 and A260/A280, respectively, using a spectrophotometer. RNA sequencing library of each sample was prepared using a TruSeq RNA Library Prep Kit. RNA-seq experiments were performed for hADSCs. Cells were transfected with either GSTT1 at 100 nM or siControl (scramble control) by electroporation using a 1050 pulse voltage for 30 ms with 2 pulses using a 10 μl pipette tip.

The purpose of this study is to discover genetic markers that can promote osteogenic differentiation of adipose stem cells (hADSCs) through mRNA-seq gene analysis. The selected GSTT1 gene was found to be associated with the enhancement of osteogenic differentiation of adipose stem cells. siRNA against GSTT1 reduced osteogenic differentiation of hADSCs, whereas GSTT1 overexpression enhanced osteogenic differentiation of hADSCs under osteogenic conditions.

In this study, GSTT1 transgenic adipose stem cells could be used in regenerative medicine to improve bone differentiation. In addition, the GSTT1 gene has important significance as a marker for selecting adipose stem cells with potential for bone differentiation in the development of a therapeutic agent for bone regeneration cells.

Stem cells are known to have low levels of intracellular reactive oxygen species (ROS) and high levels of glutathione. ROS are thought to interact with several pathways that affect the transcription machinery required for stem cell differentiation, and are critical for maintaining stem cell function. In this study, we are developing a new fluorescent probe that rapidly and reversibly reacts with glutathione (GSH), the most abundant non-protein thiol in living cells that acts as an antioxidant and redox regulator.

Multipotent perivascular progenitor cells derived from human ESCs (hESC-PVPCs): Differentiated ESCs as embryoid bodies in the presence of BMP4 to induce mesoderm differentiation followed by a simple cell selection strategy using attachment of single cells onto collagen-coated dishes. Differential gene expression profiling was performed among H9 hESCs, EBs induced by BMP4 and naturally selected CD140B+CD44+ population at Day 7 (PVPCs). Colony-forming assay: GSHhigh and GSHlow PVPCs were plated on 10-cm tissue culture-treated polystyrene dishes in triplicate in growth medium and cultured for 14 days. Transwell migration assay: GSHhigh and GSHlow PVPCs at passage 4 were resuspended at 1 × 10⁶/mL in the migration medium and seeded in the upper chamber. The following human recombinant SDF-1 and PDGF-AA proteins were used as chemoattractants in the lower compartment.

Probe-GSH conjugate shows shifts in fluorescence excitation and emission spectra that enables ratiometric measurement of GSH levels. Using these properties, stem cells can be purified by FACS-based technology according to intracellular GSH level. We are developing a protocol both for comparing GSH level in stem cell from different culture conditions and for preparing stem cells with high-GSH level . Our results reveal that GSHhigh PVPC purified by FACS show increased colony forming ability compared with that GSHlow PVPC, indicating that intracellular GSH contributes to the maintenance of stemness. Moreover, transplantation of GSHlow PVPC is more effective than that of GSHlow PVPC for cartilage regeneration in osteochondral defect.

This technique enable FACS-based sorting of stem cells according to intracellular GSH levels and thus investigation of functional role of GSH (high antioxidant capacity) in the stem cell maintenance and chondrogenic differentiation.

Mesenchymal stem cells (MSC) have been used for bone regenerative applications as an alternative approach to bone grafting. Selecting the appropriate source of MSC is vital for the success of this therapeutic approach. MSC can be obtained from various tissues, but the most used sources of MSC are Bone marrow (BMSC), followed by adipose tissue (ASC). A donor-matched comparison of these two sources of MSC ensures robust and reliable results.

Despite the similarities in morphology and immunophenotype of donor-matched ASC and BMSC, differences existed in their proliferation and in vitro differentiation potential, particularly osteogenic differentiation that was superior for BMSC, compared to ASC. However, these differences were substantially influenced by donor variations. In vivo, although the upregulated expression of osteogenesis-related genes in both ASC and BMSC, more bone was regenerated in the calvarial defects treated with BMSC compared to ASC, especially during the initial period of healing. According to these findings, compared to ASC, BMSC may result in faster regeneration and healing, when used for bone regenerative applications.

Introduction: Emerging therapies for regenerating skeletal tissues are focused on the repair of pathologically altered tissue by the transplantation of functionally competent cells and supportive matrices. Stem cells have the potential to differentiate into musculoskeletal tissue and may be the optimal cell source for such therapies. In vitro studies have demonstrated the ability of adult bone marrow stromal cells (MSC) and human embryonic stem cells (hES) to generate bone, but little is known regarding their potential to repair bone in vivo. Preclinical studies in animal models will allow investigation into the extent that regenerated tissue resembles functional and healthy tissue, and its potential clinical application. Aim: To assess whether adult and embryonic stem cells maintained their ability to form musculoskeletal tissues in vivo using diffusion chambers implanted into the peritoneal cavity of nude mice. Currently, ongoing experiments are assessing the use of MSCs and hES cells to regenerate bone in a rodent preclinical model. Methods: MSC cells and embryoid body-derived H9 hES cells were prepared as previously described (Haynesworth et al Bone 1992; Sottile et al Cloning Stem Cells 2003). Groups of cells were left untreated or pre-treated with osteogenic (OS) media for 5 days. Study 1: Single cell suspensions of untreated or pre-treated cells were injected into diffusion chambers which were implanted intraperitonealy into nude mice and left for 79 days. Study 2: OS pre-treated cells were implanted into an experimentally created full thickness calvarial defect in adult male Wistar rats. The defect area was left empty or filled with demineralised bone matrix (DBM: Allosource®) alone or with DBM/MSCs or DBM/hES composite. Tissues were collected 4 weeks after surgery. Analysis: Histological and immunochemical techniques were used to evaluate cell phenotypes and the contribution of transplanted cells to tissue repair. Results: Study 1: Both hES (in 2/3 chambers) and MSC (3/3) cells pre-treated with OS media formed only mineralised bone. No cartilage was detected in these OS pre-treated cells. Untreated hES cells formed both mineralised bone and cartilage within the chambers (2/3). In contrast, untreated MSC cells (3/3) produced no mineralised bone or cartilage. Preliminary analysis demonstrated the absence of any other tissue type in the diffusion chambers. Study 2: Active bone regeneration was observed at the edges of the calvarial defect after 4 weeks, with a high density of cells present within the MSC or hES/DBM composite. No signs of local cellular immunological response were seen. Summary: OS pre-treatment restricted differentiation towards the osteoblast lineage in both hES and MSC cells indicating successful directed differentiation in vivo. Untreated hES and MSC cells produce a different range of cell phenotypes suggesting that the two cell sources represent cells at a different stage of commitment in a common cell lineage or cells derived from two distinct cell lineages. New bone formation was seen at the site of the calvarial defect in the presence OS pre-treated MSC and hES cells suggesting that these cells may support in vivo bone repair in a preclinical model. Funded by Geron Corporation

A promising application of Mesenchymal stem cells (MSCs) is the treatment of non-unions. Substituting bone grafts, MSCs are directly injected into the fracture gap. High cell viability seems to be a prerequisite for therapeutic success. Administration of the MSCs via injection creates shear stresses possibly damaging or destroying the cells.

Aim of this study was to investigate the effect of the injection process on cell viability.

MSCs were isolated and cultivated from femoral tissue of five subjects undergoing arthroplasty. Prior to injection, the cells were identified as MSCs. After dissolving to a concentration of 1 Million cells/ml, 1 ml of the suspension was injected through a cannula of 200 mm length and 2 mm diameter (14 G) with flow rates of 38 and 100 ml/min. The viability of the MSCs at different flow rates was evaluated by staining to detect the healthy cell fraction. It was analyzed statistically against a control group via the Kruskal-Wallis-test and for equivalence via the TOST procedure. Significance level was set to 5 %, equivalence margin to 20 %.

The healthy cell fraction of the control group was 85.88 ± 2.98 %, 86.04 ± 2.53 % at 38 ml/min and 85.48 ± 1.64 % at 100 ml/min. There was no significant difference between the fraction of healthy cells (p = 0.99) for different volume flows, but a significant equivalence between the control group and the two volume flows (38 ml/min: p = 0.002, 100 ml/min: p = 0.001).

When injecting MSC solutions, e.g. into a non-union, the viability of the injected cells does not deterioriate significant. The injecting technique is therefore feasible.

Background: It has been previously shown that in elderly patients with osteoporosis the Mesenchymal Stem Cell (MSC) growth rate and osteogenic potential is decreased. The aim of this study was to elucidate the effect of BMP-2, BMP-7, PTH and PDGF on MSC’s capacity to proliferate and differentiate. Methods: Cancellous bone samples were obtained from 10 patients (mean age 76 (70–84), (4 males)) suffering from lower extremity fractures and osteoporosis. Mes-enchymal Stem Cells (MSCs) were isolated by enzymatic digestion. Cells were cultured till passage 3 (P3). Functional assays on proliferation and osteogenic differentiation were performed under the influence of a wide range of BMP-2, BMP-7, PTH and PDGF concentrations. Proliferation was assessed using CFU-F and XTT assays. Osteogenic differentiation was assessed by alkaline phosphatase activity and total calcium production. Results: MSC proliferation was found upregulated by medium supplementation with BMP-7 and PDGF. The highest proliferation rate increase was achieved with 100 ng/ml of BMP-7. BMP-2 and PTH did not affect MSC proliferation. All four molecules upregulated ALP activity and calcium production by growing osteoblasts. A dose dependant effect was noted. BMP-2 and BMP-7 in their highest studied concentration (100 ng/ml) produced a ~ three-fold increase on osteogenic potential of MSCs. Conclusion: This study indicates that BMP-7 and BMP-2 have favourable effect on osteogenic differentiation of MSCs. However, BMP-7 could be more advantageous as it enhances both proliferation and osteogenic differentiation of MSCs derived from elderly osteoporotic bone

To test and evaluate the effectiveness of local injection of autologous fat-derived mesenchymal stem cells (MSCs) into fracture site to prevent non-union in a clinically relevant model.

5 male Wistar rats underwent the same surgical procedure of inducing non-union. A mid-shaft tibial osteotomy was made with 1mm non-critical gap. Periosteum was stripped around the two fracture ends. Then, the fracture was fixed by ante-grade intramedullary nail. The non-critical gap was maintained by a spacer with minimal effect on the healing surface area. At the same surgical time, subcutaneous fat was collected from the ipsilateral inguinal region and stem cells were isolated and cultured in vitro. Within three weeks postoperatively, the number of expanded stem cells reached 5×10⁶ and were injected into the fracture site. Healing was followed up for 8 weeks and the quality was measured by serial x-rays, microCT, mechanical testing and histologically. Quality of healing was compared with that of previously published allogenic, xenogeneic MSCs and Purified Buffered Saline (PBS) controls.

All the five fractures united fully after 8 weeks. There was a progressive increase in the callus radiopacity during the eight-week duration, the average radiopacity in the autologous fat-MSC injected group was significantly higher than that of the allogeneic MSCs, xenogeneic MSCs and the control group, P < 0.0001 for treatment, time after injection, and treatment-time interaction (two-way repeated measure ANOVA). MicroCT, mechanical testing and histology confirmed radiological findings.

The autologous fat-MSCs are effective in prevention of atrophic non-union by stimulation of the healing process leading to a solid union. The quality and speed of repair are higher than those of the other types of cell transplantation tested.

Aneurysmal bone cyst (ABC) of the spine is a locally aggressive benign lesion which can be treated by en bloc resection with wide margin to reduce the risk of local recurrence. To avoid morbidity associated with surgery, selective arterial embolization (SAE) can be considered the first-line treatment for ABCs of the spine. We previously introduced the use of autologous bone marrow concentrate (BMC) injection therapy to stimulate bone healing and regeneration in ABC of the spine. In this prospective study we described the clinical and radiological outcomes of percutaneous injection of autologous BMC in a series of patients affected by ABCs of the spine.

Fourteen patients (6 male, 8 female) were treated between June 2014 and December 2019 with BMC injection for ABC of the spine. The mean age was 17.85 years. The mean follow up was 37.4 months (range 12–60 months). The dimension of the cyst and the degree of ossification were measured by Computed Tomography (CT) scans before the treatment and during follow-up visits.

Six patients received a single dose of BMC, five patients received two doses and in three patients three doses of BMC were administered. The mean ossification of the cyst (expressed in Hounsfield units) increased statistically from 43.48±2.36 HU to 161.71±23.48 HU during follow-up time and the ossification was associated to an improvement of the clinical outcomes. The mean ossification over time was significantly higher in patients treated with a single injection compared to patients treated with multiple injections. No significant difference in ossification was found between cervical and non-cervical localization of the cyst. Moreover, the initial size of the cyst was not statistically associated with the degree of ossification during follow-up.

The results of this study reinforce our previous evidence on the use of BMC as a valid alternative for spinal ABC management when SAE is contraindicated or ineffective.

The initial size of the cyst and its localization does not influence the efficacy of the treatment. However, data suggest that BMC injection could be indicated as treatment of choice for spinal ABC in young adolescent women.

Extensive bone defects, caused by severe trauma or resection of large bone tumors, are difficult to treat. Regenerative medicine, including stem cell transplantation, may provide a novel solution for these intractable problems and improve the quality of life in affected patients. Adipose-derived stromal/stem cells (ASCs) have been extensively studied as cell sources for regenerative medicine due to their excellent proliferative capacity and the ability to obtain a large number of cells with minimal donor morbidity. However, the osteogenic potential of ASCs is lower than that of bone marrow-derived stromal/stem cells. To address this disadvantage, our group has employed various methods to enhance osteogenic differentiation of ASCs, including factors such as bone morphogenetic protein or Vitamin D, coculture with bone marrow stem cells, VEGF transfection, and gene transfer of Runx-2 and osterix. Recently, we mined a marker that can predict the osteogenic potential of ASC clones and also investigated the usefulness of the molecule as the enhancer of osteogenic differentiation of ASCs as well as its mechanism of action. Through RNA-seq gene analysis, we discovered that GSTT1 was the most distinguished gene marker between highly osteogenic and poorly osteogenic ASC clones. Knockdown of GSTT1 in high osteogenic ASCs by siGSTT1 treatment reduced mineralized matrix formation while GSTT1 overexpression by GSTT1 transfection or GSTT1 recombinant protein treatment enhanced osteogenic differentiation of low osteogenic ASCs. Metabolomic analysis confirmed significant changes of metabolites related to bone differentiation in ASCs transfected with GSTT1. A high total antioxidant capacity, low levels of cellular reactive oxygen species and increased GSH/GSSG ratios were also detected in GSTT1- transfected ASCs. GSTT1 can be a useful marker to screen the highly osteogenic ASC clones and also a therapeutic factor to enhance the osteogenic differentiation of poorly osteogenic ASC clones.

In this study, we developed biocompatible adhesive which enables implanted chondrogenic-enhanced hASCs being strongly fixed to the lesion site of defected cartilage.

The bioengineered mussel adhesive protein (MAP) was produced and purified using a bacterial expression system as previously reported. The cell encapsulated coacervate was formulated with two polyelectrolyte, the MAP and 723kDa hyaluronic acid (HA). MAP formed liquid microdroplets with HA and subsequently gelated into microparticles, which is highly viscous and strongly adhesive.

The MAP with chondro-induced hASCs were implanted on the osteochondral defect created in the patellar groove/condyle of OA-induced rabbits. Rabbits were allocated to three different groups as follows: Group1 – Fibrin only; Group2 – Fibrin with hASCs (1.5×10⁶ chondro-induced hASCs); Group3; MAP with hASCs.

The implanted cells were labeled with a fluorescent dye for in vivo visualization. After 35 days, fluorescent signals were more potently detected for MAP with hASCs group than Fibrin with hASCs group in osteochondral defect model. Moreover, histological assessment showed that MAP with hASCs group had the best healing and covered with hyaline cartilage-like tissue. The staining image shows that MAP with hASCs group were filled with perfectly differentiated chondrocytes. Although Fibrin with hASCs group had better healing than fibrin only group, it was filled with fibrous cartilage which owes its flexibility and toughness. As MAP with hASCs group has higher possibility of differentiating to complete cartilage, Fibrin only group and Fibrin with hASCs group have failed to treat OA by rehabilitating cartilage. In order to clarify the evidence of remaining human cell proving efficacy of newly developed bioadhesive, human nuclear staining was proceeded with sectioned rabbit cartilage tissue. The results explicitly showed MAP with hASCs group have retained more human cells than Fibrin only and Fibrin with hASCs groups.

We investigated the waterproof bioadhesive supporting transplanted cells to attach to defect lengthily in harsh environment, which prevents cells from leaked to other region of cartilage. Collectively, the newly developed bio-adhesive, MAP, could be successfully applied in OA treatment as a waterproof bioadhesive with the capability of the strong adhesion to target defect sites.

Numerous investigators have described osteogenic differentiation of bone marrow stromal cells obtained from both murine and human sources over the past decade. The ease of access and large available quantity of adipose tissue, however, makes Adipose-Derived Stem Cells (ADSC) a far more practical alternative for clinical applications, such as operative treatment of non-unions and regeneration of critical bone defects. Therefore, the primary goal of this research endeavor is to achieve osteogenic differentiation of ADSC. Previous work has already demonstrated that bone morphogenetic protein receptor 1A (BMP receptor 1A) signaling is required for healing critical bone defects. Based on this evidence, we used a lentiviral vector to increase expression of BMP receptor 1A by our stem cell population in order to direct their differentiation into the osteoblastic lineage. We harvested subcutaneous adipose tissue intraoperatively from consenting patients undergoing elective lipoplasty and panniculectomy procedures. The stromal vascular fraction was isolated from this tissue and further refined by passaging in selective media to yield a stable population of ADSC in primary culture. Both the identity and homogeneity of this stem cell population was confirmed using adipogenic induction media and differentiation cocktails. In addition, we subcloned an expression plasmid containing the BMP receptor 1A locus in tandem with green fluorescent protein (GFP) under the transcriptional control of a single promoter. This plasmid was packaged into a lentiviral vector to provide a reliable method of achieving both genomic integration and long-term expression of the BMP receptor 1A gene. Hence, transduction of ADSC using this vector resulted in overexpression of BMP receptor 1A by these multipotent cells. The GFP was then utilized as a reporter gene to screen and enrich the ADSC population for only those stem cells with a robust expression of BMP receptor 1A. The ADSC that overexpressed BMP receptor 1A were found to achieve osteogenic differentiation after 18 to 20 days of in vitro culture, as revealed by immunohistochemistry assays for osteocalcin. Osteogenic differentiation was further confirmed by alizarin red staining and quantitative PCR for alkaline phosphatase gene expression as a biomarker for the osteoblastic lineage. Our results demonstrate that stem cells derived from the adipose tissue of a patient represent a viable means of culturing autologous osteoblasts in vitro for future implantation at the site of critical bone defects. This method of attaining osseous regeneration is intuitively appealing, given the minimal donor site morbidity associated with removing subcutaneous fat. By transducing the ADSC with a lentiviral vector, we have also collected further evidence implicating the critical importance of signaling mediated by the BMP receptor 1A during osteogenesis. Further tissue engineering studies are now in progress to evaluate the osteogenic differentiation potential of these stem cells under hydrostatic and fluid flow shearing mechanical loads

Purpose: Athrophic non unions constitute a major problem in orthopaedic trauma. The main probably cause of atrophic non union is damage of the vascular system and dysfunctional regeneration of the vasculature at the area of the fracture. The most important hormonal pathway controlling angiogenesis is VEGF (Vascular Endothelial Growth Factor). The use of VEGF for enhancing bone healing in atrophic non unions could be a very promising solution for the future. An interesting alternative to the use of VEGF is the use of Erythropoietin (Epo). VEGF has been also reported to interact with Endothelial Progenitor Cells (EPCs). Our scope is to identify a possible new role for Epo as a valid substitute for VEGF through the clarification of the molecular and cellular pathways of fracture healing. Methods: A survey was conducted via internet (Med-line - Pubmed, Cochrane database, Scopus) and relevant textbooks. Results: It has been reported that Epo could induce increased chemotaxis, migration of Mesenchymal Stem Cells (MSCs), but also activation of Metaloproteinase - 9 and production of pro-angiogenic factors. These effects on MSCs could explain the observation that Epo could be very useful in the treatment of wound healing and burn healing in animal studies. It has been that Epo could express receptors at the chondrocytes, but also induce better bio-mechanical strength, callus formation, histomophometric image and increased bone density at the treated with Epo animals when compared with control animals. It is also worthy to note that the Epo has been found to stimulate neo-vascularisation in vivo, differentiation of endothelial cell lines towards a vascular pathway and improvement of cardiac function through EPCs and VEGF, implying Epo also in the differentiation and chemotaxis of the circulating EPCs. We should not forget that the transformation of EPCs in mesenchymal cells (i. g. myoblasts) has already been demonstrated. Conclusions: The consequences of these observations could be very interesting: EPCs have been reported to enhance neo-vascularisation and angiogenesis at the region of the fracture. All these imply a novel role for EPCs in combination (or even replacing the rare) MSCs under the influence of VEGF and Epo for the enhancement of fracture vascularisation and healing enhancement. Further studies should clarify this new field in basic orthopaedic, trauma and bone metabolism science

Numerous investigators have described chondrogenic differentiation of bone marrow stromal cells obtained from both murine and human sources over the past decade. The ease of access and large available quantity of adipose tissue, however, makes Adipose-Derived Stem Cells (ADSC) a far more practical alternative for clinical applications. Therefore, the primary goal of this research endeavor is to achieve chondrogenic differentiation of ADSC. Previous work had also demonstrated that bone morphogenetic protein receptor 1A (BMP receptor 1A) signaling is required for postnatal maintenance of articular cartilage. In fact, cartilage within the joints of transgenic mice deficient in BMP receptor 1A rapidly degenerates after birth in a process resembling accelerated human osteoarthritis. Based on this evidence, we used a lentiviral vector to increase expression of BMP receptor 1A by our isolated stem cells in order to direct their differentiation into the chondrocyte lineage. We harvested subcutaneous adipose tissue intraoperatively from consenting patients undergoing elective lipoplasty and panniculectomy procedures. The stromal vascular fraction was isolated from this tissue and further refined by passaging in selective media to yield a stable population of ADSC in primary culture. Both the identity and homogeneity of this stem cell population was confirmed using adipogenic induction media and differentiation cocktails. In addition, we subcloned an expression plasmid containing the BMP receptor 1A locus in tandem with green fluorescent protein (GFP) under the transcriptional control of a single promoter. This plasmid was packaged into a lentiviral vector to provide a reliable method of achieving both genomic integration and long-term expression of the BMP receptor 1A gene. Hence, transduction of ADSC using this vector resulted in overexpression of BMP receptor 1A by these multipotent cells. The GFP was then utilized to screen and enrich the ADSC population for stem cells with a robust expression of BMP receptor 1A. The ADSC that overexpressed BMP receptor 1A were found to achieve chondrogenic differentiation after 13 to 16 days of in vitro culture, as revealed by immunohistochemistry assays for the bio-markers of articular cartilage (type II collagen and the proteoglycan aggrecan). Our results demonstrate that stem cells derived from the adipose tissue of a patient represent a viable means of culturing autologous chondrocytes in vitro for future implantation at the site of osteochondral defects. This method of attaining cartilaginous regeneration is intuitively appealing, given the minimal donor site morbidity associated with removing subcutaneous fat. By transducing the ADSC with a lentiviral vector, we have also collected further evidence implicating the critical importance during chondrogenesis of signaling mediated by the BMP receptor 1A. Further tissue engineering studies are now in progress to evaluate the ability of ADSC to differentiate into chondrocytes after seeding onto poly-caprolactone polymer scaffolds