Advanced Therapeutic Medicinal Products in Bone and Cartilage Defects.

The number of patients with functional loss of bone and cartilage tissue has shown an increasing trend. Insufficient or inappropriate conventional treatments applied for trauma, orthopedic diseases, or other bone and cartilage-related disorders can lead to bone and cartilage damage. This represents a worldwide public health issue and a significant economic burden. Advanced therapeutic medicinal products (ATMPs) proposed promising alternative therapeutic modalities by application of cell-based and tissue engineering approaches. Recently, several ATMPs have been developed to promote bone and cartilage tissue regeneration. Fifteen ATMPs, two related to bone and 13 related to cartilage, have received regulatory approval and marketing authorization. However, four ATMPs were withdrawn from the market for various reasons. However, ATMPs that are still on the market have demonstrated positive results, their broad application faced limitations. The development and standardization of methodologies will be a major challenge in the coming decades. Currently, the number of ATMPs in clinical trials using mesenchymal stromal cells or chondrocytes indicates a growing recognition that current ATMPs can be improved. Research on bone and cartilage tissue regeneration continues to expand. Cell-based therapies are likely to be clinically supported by the new ATMPs, innovative fabrication processes, and enhanced surgical approaches. In this study, we highlighted the available ATMPs that have been used in bone and cartilage defects and discussed their advantages and disadvantages in clinical applications.

Allogeneic Bone Marrow Mesenchymal Stromal Cell Transplantation Induces Dentin Pulp Complex-like Formation in Immature Teeth with Pulp Necrosis and Apical Periodontitis.

INTRODUCTION: Dental pulp regeneration is challenging in endodontics. Cellular therapy is an alternative approach to induce dental pulp regeneration. Mesenchymal stromal cells (MSCs) have the capacity to induce dental pulp-like tissue formation. In this study, we evaluated the capacity of allogeneic bone marrow MSCs (BM-MSCs) to regenerate pulp following necrosis and apical periodontitis in children's permanent immature apex teeth. METHODS: Patients aged 8 to 12 years with pulp necrosis and apical periodontitis were evaluated. The study included 15 teeth (13 incisors and 2 molars) from 14 patients (8 boys and 6 girls). Radiographic evaluation showed periapical radiolucency and immature apex teeth. There was no response to cold or electric pulp testing. The root canal of each tooth was cleaned, shaped, and Ca(OH)2 used as an interappointment medication. Cryopreserved allogeneic BM-MSCs were thawed, expanded, incorporated into preclotted platelet-rich plasma, and implanted into the tooth's pulp cavity. They were sealed with bioceramic cement and composite. Sensibility, apical foramen, calcium deposits within the root canal, and resolution of periapical lesions were evaluated in each tooth over the following 12 months. RESULTS: Based on 9 variables established for dental pulp-like tissue regeneration, all MSC-treated teeth showed evidence of successful regeneration. Clinical and radiographic evaluation of the treated teeth showed periapical lesion healing, sensitivity to cold and electricity, decreased width of the apical foramen, and mineralization within the canal space. CONCLUSIONS: Transplantation of allogeneic MSCs induces the formation of dental pulp-like tissue in permanent immature apex teeth with pulp necrosis and apical periodontitis. Implant of MSCs constitutes a potential therapy in regenerative endodontics in pediatric dentistry. Future studies incorporating a larger sample size may confirm these results.

Osteogenic organoid for bone regeneration: Healing of bone defect in congenital pseudoarthrosis of the tibia.

BACKGROUND: Congenital pseudoarthrosis of the tibia (CPT) is an uncommon disease associated with failure to achieve bone union and recurrent fractures. There is evidence showing that CPT is associated with decreased osteogenesis. Based on the capacity of mesenchymal stromal cells (MSCs) to induce osteogenesis, we develop an osteogenic organoid (OstO) constituted by these cells, and other components of the bone niche, for inducing bone formation in a child diagnosed with CPT. AIM: To evaluate the capacity of an OstO to induce bone formation in a patient with CPT. METHODS: The OstO was fabricated with allogeneic bone marrow MSCs from a healthy donor, collagen microbeads (CM) and PRP clot. The CM and PRP function as extracellular matrix and scaffolds for MSC. The OstO was placed at the site of non-union. Internal and external fixation was placed in the tibia. Radiological evaluation was performed after MSCs transplantation. RESULTS: After 4 months of MSCs transplantation, radiographic imaging showed evidence of osteogenesis at the site of CPT lesion. The tibia showed bone consolidation and complete healing of the non-union CPT lesion after 6 months. Functional improvement was observed after 1 year of MSC transplantation. CONCLUSIONS: The OstO is a bone-like niche which promote osteogenesis in patients with failure in bone formation, such as CPT. To our knowledge, these results provide the first evidence showing CPT healing induced by an OstO constituted by allogeneic MSCs. Future studies incorporating a larger number of patients may confirm these results.

Cartilage regeneration and inflammation modulation in knee osteoarthritis following injection of allogeneic adipose-derived mesenchymal stromal cells: a phase II, triple-blinded, placebo controlled, randomized trial.

BACKGROUND: Intra-articular injection of mesenchymal stromal cells (MSCs) with immunomodulatory features and their paracrine secretion of regenerative factors proposed a noninvasive therapeutic modality for cartilage regeneration in knee osteoarthritis (KOA). METHODS: Total number of 40 patients with KOA enrolled in two groups. Twenty patients received intra-articular injection of 100 × 106 allogeneic adipose-derived mesenchymal stromal cells (AD-MSCs), and 20 patients as control group received placebo (normal saline). Questionnaire-based measurements, certain serum biomarkers, and some cell surface markers were evaluated for 1 year. Magnetic resonance imaging (MRI) before and 1 year after injection was performed to measure possible changes in the articular cartilage. RESULTS: Forty patients allocated including 4 men (10%) and 36 women (90%) with average age of 56.1 ± 7.2 years in control group and 52.8 ± 7.5 years in AD-MSCs group. Four patients (two patients from AD-MSCs group and two patients from the control group) excluded during the study. Clinical outcome measures showed improvement in AD-MSCs group. Hyaluronic acid and cartilage oligomeric matrix protein levels in blood serum decreased significantly in patients who received AD-MSCs (P < 0.05). Although IL-10 level significantly increased after 1 week (P < 0.05), the serum level of inflammatory markers dramatically decreased after 3 months (P < 0.001). Expressions of CD3, CD4, and CD8 have a decreasing trend during 6-month follow-up (P < 0.05), (P < 0.001), and (P < 0.001), respectively. However, the number of CD25+ cells increased remarkably in the treatment group 3 months after intervention (P < 0.005). MRI findings showed a slight increase in the thickness of tibial and femoral articular cartilages in AD-MSCs group. The changes were significant in the medial posterior and medial anterior areas of ​​the tibia with P < 0.01 and P < 0.05, respectively. CONCLUSION: Inter-articular injection of AD-MSCs in patients with KOA is safe. Laboratory data, MRI findings, and clinical examination of patients at different time points showed notable articular cartilage regeneration and significant improvement in the treatment group. TRIAL REGISTRATION: Iranian registry of clinical trials (IRCT, https://en.irct.ir/trial/46 ), IRCT20080728001031N23. Registered 24 April 2018.

Wound healing by transplantation of mesenchymal stromal cells loaded on polyethylene terephthalate scaffold: Implications for skin injury treatment.

BACKGROUND: Several clinical studies have shown that cellular therapy based on mesenchymal stromal cells (MSCs) transplantation may accelerate wound healing. One major challenge is the delivery system used for MSCs transplantation. In this work, we evaluated the capacity of a scaffold based on polyethylene terephthalate (PET) to maintain the viability and biological functions of MSCs, in vitro. We examined the capacity of MSCs loaded on PET (MSCs/PET) to induce wound healing in an experimental model of full-thickness wound. METHODS: Human MSCs were seeded and cultured on PET membranes at 37 °C for 48 h. Adhesion, viability, proliferation, migration, multipotential differentiation and chemokine production were evaluated in cultures of MSCs/PET. The possible therapeutic effect of MSCs/PET on the re-epithelialization of full thickness wounds was examined at day 3 post-wounding in C57BL/6 mice. Histological and immunohistochemical (IH) studies were performed to evaluate wound re-epithelialization and the presence of epithelial progenitor cells (EPC). As controls, wounds without treatment or treated with PET were established. RESULTS: We observed MSCs adhered to PET membranes and maintained their viability, proliferation and migration. They preserved their multipotential capacity of differentiation and ability of chemokine production. MSCs/PET implants promoted an accelerated wound re-epithelialization, after three days post-wounding. It was associated with the presence of EPC Lgr6+ and K6+. DISCUSSION: Our results show that MSCs/PET implants induce a rapid re-epithelialization of deep- and full-thickness wounds. MSCs/PET implants constitute a potential clinical therapy for treating cutaneous wounds.

3D organoid modeling of extramedullary hematopoiesis.

BACKGROUND: Under certain clinical and experimental conditions hematopoiesis occurs in other site than bone marrow (BM), such as the liver. Here, we develop a 3D organoid that mimics several components of the hematopoietic niche present during liver extramedullary hematopoiesis. AIM: To evaluate the capacity of a 3D hematopoietic organoid (3D-HO) to function as a hematopoietic like-niche allowing for blood cell production outside of the BM. METHODS: The 3D-HO is constituted by liver sinusoidal endothelial cells (LSEC) as the stromal component, BM isolated from 5-FU treated mice (FU-BMCs), collagen microspheres and plasma clot as scaffolds. The ability of the 3D-HO to support the survival and functionality of FU-BMCs was investigated by using confocal microscopy, histology analysis, flow cytometry, and clonogenic assays. RESULTS: After 15 and 30 days, post-ectopic implantation, histological studies of the 3D-HO showed the presence of cells with myeloid and lymphoid lineage morphology. Flow cytometry analysis of these cells showed the presence of cells expressing hematopoietic stem progenitor cells (HSPC) (Sca-1+/c-Kit+), myeloid (Gr-1+) and lymphoid (B220+ and CD19+) markers. Clonogenic assays showed that cells from the 3D-HO formed hematopoietic colonies. Expression of the Sry gene by cells from the 3D-HO, implanted for 30 days in female mice, indicated that male donor cells persist in this model of extramedullary hematopoiesis. CONCLUSIONS: The 3D-HO constitutes an extramedullary hematopoietic-like niche which supports the survival and functionality of FU-BMCs. It may constitute an efficient model for investigating, in vitro and in vivo, those factors that control hematopoiesis outside BM.

The effects of mesenchymal stromal cells and platelet-rich plasma treatments on cutaneous wound healing.

Cellular therapy and platelet-rich plasma (PRP) have been used as a treatment for skin wounds. Previous evidence has shown that mesenchymal stromal cells (MSC) may improve skin wound healing. In contrast, contradictory effects have been reported by using PRP treatment on skin wound healing. However, there is evidence that PRP constitutes an excellent scaffold for tissue engineering. In this work, we aim to study the effect of MSC on skin wound healing. We used an experimental murine model of full-thickness wounds. Wounds were treated with human bone marrow-MSC contained in a PRP clot. Untreated or PRP-treated wounds were used as controls. Wound healing was evaluated by macroscopic observation and histological analysis at day 7 post-wounding. Immunohistochemical studies were performed to detect the presence of epithelial progenitor cells (EPC) and the expression of basic fibroblast growth factor (bFGF). MSC/PRP implantation induced a significant wound closure and re-epithelialization as compared with the controls. Increase of CD34+ cells and bFGF was observed in the wounds treated with MSC/PRP. Our results show that MSC included in PRP clot induce cutaneous wound repair by promoting re-epithelialization, migration of EPC and expression of bFGF. PRP alone does not exert a significant effect on wound healing. Our results support the possible clinical use of MSC in PRP scaffold as potential treatment of skin wounds.

Dental Pulp Regeneration Induced by Allogenic Mesenchymal Stromal Cell Transplantation in a Mature Tooth: A Case Report.

INTRODUCTION: Cellular therapy constitutes a new therapeutic alternative in regenerative endodontics. In this case report, we evaluated the capacity of allogeneic mesenchymal stromal cells (MSCs) to induce dental pulp and apical bone regeneration in a tooth previously endodontically treated. METHODS: A healthy 55-year-old female patient consulting for swelling and a sinus tract associated with tooth #8 was referred for an endodontic evaluation. Previously, tooth #8 had undergone root canal treatment and apical resection and had no response to thermal or electric pulp testing. Radiographically, tooth #8 showed root canal treatment, a cut apex angle, and periapical radiolucency. The root canal was recleaned and shaped, and calcium hydroxide was used as an interappointment medication. Cryopreserved allogeneic bone marrow MSCs were thawed, expanded, incorporated into preclotted platelet-rich plasma, and implanted into the pulp cavity of tooth #8. The cervical part of the canal was sealed with bioceramic cement and a composite. RESULTS: After 14 months of MSC transplantation, tooth #8 showed sensitivity to cold and electric pulp tests. Radiographic and cone-beam computed tomographic imaging showed signs of increased periapical bone density, healing of the periapical lesion, and almost complete apical remodeling. CONCLUSIONS: This case report shows periodontal bone formation, apex remodeling, and dental pulp regeneration induced by allogeneic MSC transplantation in a mature nonvital tooth. Allogeneic MSCs may constitute a first-line therapy in regenerative endodontics.

Enhanced chondrogenesis from chondrocytes co-cultured on mesenchymal stromal cells: Implication for cartilage repair.

Cellular therapy based on chondrocytes implantation is the most widely used procedure for inducing cartilage regeneration. However, the dedifferentiation process that these cells suffer and their limited capacity of proliferation, when they are cultured in vitro, restrict their use in cellular therapy protocols. To investigate the capacity of mesenchymal stromal cells (MSCs) to promote chondrogenesis from chondrocytes or chondrons in 2D and 3D coculture systems. Murine chondrocytes and chondrons were cocultured with MSCs at different cell ratios (100/0, 50/50, 70/30, 0/100) in two-dimensional (2D) and three-dimensional (3D) culture systems. High proliferation of cells with chondrocyte morphology, enhanced GAG production and expression of cartilage genes (aggrecan, type II collagen, and SOX-9) were observed in chondrocytes/MSCs cocultures. In contrast, fibroblastoid cells, down-regulation of cartilage gene expression and reduction of GAG production were observed in chondrons/MSCs cocultures. Chondrocytes within cartilage lacunae and surrounded by extracellular matrix were observed in chondrocytes/MSC pellets. MSCs promote the proliferation of functional chondrocytes in 2D and 3D culture systems. Transplantation of chondrogenic construct based on MSCs and chondrocytes may constitute a potential treatment for inducing cartilage repair.

Mesenchymal Stromal Cell Transplantation Induces Regeneration of Large and Full-Thickness Cartilage Defect of the Temporomandibular Joint.

OBJECTIVE: Cartilage damage (CD) in the temporomandibular joint (TMJ) continues being a major problem in maxillofacial field. Evidence suggests that cellular therapy may be used for repairing CD in the TMJ. DESIGN: A murine model of condyle CD (CCD) was generated in the TMJ to evaluate the capacity of mesenchymal stromal cells (MSCs) to induce cartilage regeneration in CCD. A large CCD was surgically created in a condyle head of the TMJ of C57BL/6 mice. Human MSC embedded into preclotted platelet-rich plasma (PRP) were placed on the surface of CCD. As controls, untreated CCD and exposed TMJ condyle (sham) were used. After 6 weeks, animals were sacrificed, and each mandibular condyle was removed and CCD healing was assessed macroscopically and histologically. RESULTS: Macroscopic observation of CCD treated with MSC showed the presence of cartilage-like tissue in the CCD site. Histological analysis showed a complete repair of the articular surface with the presence of cartilage-like tissue and subchondral bone filling the CCD area. Chondrocytes were observed into collagen and glycosaminoglycans extracellular matrix filling the repaired tissue. There was no evidence of subchondral bone sclerosis. Untreated CCD showed denudated osteochondral lesions without signs of cartilage repair. Histological analysis showed the absence of tissue formation over the CCD. CONCLUSIONS: Transplantation of MSC induces regeneration of TMJ-CCD. These results provide strong evidence to use MSC as potential treatment in patients with cartilage lesions in the TMJ.

A 3D construct based on mesenchymal stromal cells, collagen microspheres and plasma clot supports the survival, proliferation and differentiation of hematopoietic cells in vivo.

The hematopoietic niche is a specialized microenvironment that supports the survival, proliferation and differentiation of hematopoietic stem progenitor cells (HSPCs). Three-dimensional (3D) models mimicking hematopoiesis might allow in vitro and in vivo studies of the hematopoietic (HP) process. Here, we investigate the capacity of a 3D construct based on non-adherent murine bone marrow mononuclear cells (NA-BMMNCs), mesenchymal stromal cells (MSCs) and collagen microspheres (CMs), all embedded into plasma clot (PC) to support in vitro and in vivo hematopoiesis. Confocal analysis of the 3D hematopoietic construct (3D-HPC), cultured for 24 h, showed MSC lining the CM and the NA-BMMNCs closely associated with MSC. In vivo hematopoiesis was examined in 3D-HPC subcutaneously implanted in mice and harvested at different intervals. Hematopoiesis in the 3D-HPC was evaluated by histology, cell morphology, flow cytometry, confocal microscopy and hematopoietic colony formation assay. 3D-HPC implants were integrated and vascularized in the host tissue, after 3 months of implantation. Histological studies showed the presence of hematopoietic tissue with the presence of mature blood cells. Cells from 3D-HPC showed viability greater than 90%, expressed HSPCs markers, and formed hematopoietic colonies, in vitro. Confocal microscopy studies showed that MSCs adhered to the CM and NA-BMMNCs were scattered across the 3D-HPC area and in close association with MSC. In conclusion, the 3D-HPC mimics a hematopoietic niche supporting the survival, proliferation and differentiation of HSPCs, in vivo. 3D-HPC may allow evaluation of regulatory mechanisms involved in hematopoiesis.

Evaluation of epithelial progenitor cells and growth factors in a preclinical model of wound healing induced by mesenchymal stromal cells.

BACKGROUND: Skin wounds continue to be a global health problem. Several cellular therapy protocols have been used to improve and accelerate skin wound healing. Here, we evaluated the effect of transplantation of mesenchymal stromal cells (MSC) on the wound re-epithelialization process and its possible relationship with the presence of epithelial progenitor cells (EPC) and the expression of growth factors. METHODS: An experimental wound model was developed in C57BL/6 mice. Human MSCs seeded on collagen membranes (CM) were implanted on wounds. As controls, animals with wounds without treatment or treated with CM were established. Histological and immunohistochemical (IH) studies were performed at day 3 post-treatment to detect early skin wound changes associated with the presence of EPC expressing Lgr6 and CD34 markers and the expression of keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF). RESULTS: MSC transplantation enhanced skin wound re-epithelialization, as compared with controls. It was associated with an increase in Lgr6+ and CD34+ cells and the expression of KGF and bFGF in the wound bed. CONCLUSION: Our results show that cutaneous wound healing induced by MSC is associated with an increase in EPC and growth factors. These preclinical results support the possible clinical use of MSC to treat cutaneous wounds.

Healing of deep dermal burns by allogeneic mesenchymal stromal cell transplantation.

BACKGROUND: Deep dermal and full-thickness burns are not only difficult to treat, but they are also associated with significant morbidity and mortality. Recent reports have proposed the use of mesenchymal stromal cells (MSCs) for inducing tissue repair in burn injuries. OBJECTIVE: We aim to evaluate the effect of allogeneic MSC transplantation on full-thickness burns with delayed healing. MATERIAL AND METHODS: This study includes five patients with AB B/B burns. All patients received conservative treatments, including cleaning, debridement of necrotic tissue, and silver based dressing on the burn wounds. Cryopreserved allogeneic MSCs were thawed and rapidly expanded and used for application in burned patients. MSCs were implanted into preclotted platelet-rich plasma onto the surface of burn wounds. RESULTS: All treated burn wounds showed early granulation tissue and rapid re-epithelialization after MSC transplantation. Healing took between 1 and 5 months after MSC transplantation. Repair of burn wounds was associated with slight discoloration of the regenerated skin without hypertrophic scarring or contractures. CONCLUSION: Our results provide evidence of healing in deep- and full-thickness burns by allogeneic MSC transplantation. Rapid healing of burn patients, after MSC transplantation, improves their quality of life and reduces the length of hospitalization. Future studies incorporating a larger number of patients may confirm the results obtained in this work.

Viability and functionality of mesenchymal stromal cells loaded on collagen microspheres and incorporated into plasma clots for orthopaedic application: Effect of storage conditions.

BACKGROUND: There is evidence showing that human mesenchymal stromal cells (MSC) seeded on collagen microspheres (CM) and incorporated into platelet rich plasma (PRP) clots induce bone formation. For clinical trials it is very important to establish standardization of storage and shipment conditions to ensure the viability and functionality of cellular products. We investigate the effect of storage temperature and time on the viability and functionality of human MSC seeded on CM and included into PRP clots for using in the further clinical application for bone regeneration. METHODS: MSC/CM/PRP clots were stored at room temperature (RT), 4 °C and 37 °C for 12 h, 24 h and 48 h. At each period of time, MSC were evaluated for their viability and functionality. RESULTS: MSC from MSC/CM/PRP clots maintained at RT and 37 °C for 24 h showed a high viability (90%) and maintained their capacity of proliferation, migration and osteogenic differentiation. In contrast, MSC/CM/PRP maintained to 4 °C showed a significant reduction in their viability and migration capacity. MSC from MSC/CM/PRP clots maintained at RT for 24 h induce osteogenesis in the subcutaneous tissues of mice, after four months of transplantation. DISCUSSION: Our results show that MSC incorporated into CM/PRP clots and maintained at RT can be utilized in bone regeneration protocols during the first 24 h after their processing.

Enhanced opsonisation of Rhesus D-positive human red blood cells by recombinant polymeric immunoglobulin G anti-G antibodies.

BACKGROUND: Anti-RhD antibodies (anti-D) are important in the prophylaxis of haemolytic disease of the foetus and newborn (HDFN) due to RhD incompatibility. Current preparations of anti-D are sourced from hyperimmune human plasma, so its production carries a risk of disease and is dependent on donor availability. Despite the efforts to develop a monoclonal preparation with similar prophylactic properties to the plasma-derived anti-D, no such antibody is yet available. Here we studied the agglutinating, opsonic and haemolytic activities of two recombinant polymeric immunoglobulins (Ig) against the G antigen of the Rh complex. MATERIALS AND METHODS: Recombinant polymeric anti-G IgG1 (IgG1µtp) and IgG3 (IgG3µtp) were produced in vitro, purified by protein G-affinity chromatography, and analysed by gel electrophoresis. Their agglutinating, opsonic and haemolytic activities were evaluated using haemagglutination, erythrophagocytosis, and complement activation assays. RESULTS: The recombinant IgG1µtp and IgG3µtp anti-G antibodies ranged from 150,000 to 1,000,000 Da in molecular weight, indicating the formation of polymeric IgG. No complement activation or haemolytic activity was detected upon incubation of RhD-positive red-blood cells with the polymeric anti-G IgG. Both polymers were better opsonins than a prophylactic preparation of plasma-derived anti-D. DISCUSSION: The enhanced opsonic properties of the polymeric anti-G IgG1µtp and IgG3µtp could allow them to mediate the clearance of RhD-positive red blood cells from circulation more efficiently than natural or other synthetic prophylactic anti-D options. Their inability to induce complement-mediated haemolysis would be prophylactically convenient and is comparable in vitro to that of the available plasma-derived polyclonal anti-D preparations. The described properties suggest that polymeric antibodies like these (but with anti-D specificity) may be testable candidates for prophylaxis of HDFN caused by anti-D.

The effect of G-CSF and AMD3100 on mice treated with streptozotocin: Expansion of alpha-cells and partial islet protection.

Administration of streptozotocin (STZ) is one of the most used experimental models of diabetes (STZ-DT). STZ induces beta-cell damage in pancreatic islets. It is known that hematopoietic stem progenitor cells (HSPCs) are mobilized from bone marrow to damaged tissues. In this work, we evaluated the effects of the hematopoietic mobilizers G-CSF (250µg/kg; for five consecutive days) and AMD3100 (5mg/kg; single s.c injection) in mice treated with STZ (175mg/kg). Mice injected with STZ showed a significant reduction in the number and area of islets and in the number of beta- and alpha-cells. Concurrently, they had hyperglycemia (blood glucose over 300mg/dl) associated with very low levels of insulin in plasma. The number and area of islets from STZ-DT mice treated with G-CSF and/or AMD3100 were similar to the controls. However, these mice had neither a reduction of hyperglycemia nor an improvement in the insulin levels. Analysis of islet cellularity showed a large reduction in beta-cells with a significant expansion of alpha-cells. These results indicate that G-CSF and AMD3100 induce partial protection of islet tissues and expansion of alpha-cells in mice treated with STZ but do not protect beta-cells from the damage induced by this compound.

A method of treatment for nonunion after fractures using mesenchymal stromal cells loaded on collagen microspheres and incorporated into platelet-rich plasma clots.

PURPOSE: There is evidence showing that mesenchymal stromal cells (MSC) may constitute a potential therapeutic strategy to induce bone regeneration. In this work, we investigate the capacity of autologous bone marrow (BM) MSC loaded on collagen microspheres (CM) and included into autologous platelet-rich plasma (PRP) clots (MSC/CM/PRP) to induce bone formation in patients with nonunion lesions. METHODS: MSC were isolated from BM cells of patients with nonunion lesions. Phenotypical (marker expression) and functional studies (osteogenic differentiation) were performed. MSC were seeded on CM and included into autologous PRP clot (MSC/CM/PRP). The capacity of MSC/CM/PRP to induce bone formation was evaluated in three patients diagnosed with nonunion. RESULTS: MSC loaded on CM/PRP clots maintain their biological functions, in vitro. After three months, post-MSC transplantation, all patients showed evidence of osteogenesis at the site of nonunion. After one year, all patients showed a complete healing of the nonunion. CONCLUSIONS: Our results support the use of autologous MSC transplanted as MSC/CM/PRP for the treatment of nonunion fractures. Future studies incorporating a larger number of patients may confirm the results obtained in this work.

Isolation and Characterization of Multipotential Mesenchymal Stromal Cells from Congenital Pseudoarthrosis of the Tibia: Case Report.

Congenital pseudoarthrosis of the tibia (CPT) is an uncommon disease whose etiology and pathogenesis is unknown. Several evidences suggest that decreased osteogenic capacities, impaired local vascularization, and microenvironment alterations may play a role in the pathogenesis of CPT. Additionally, it is not clear if the pathogenesis of this disease is related to the absence of cells with osteogenic capacity of differentiation. In this work, a two-year-old patient diagnosed with CPT underwent an orthopedic surgery to promote bone union in a pseudoarthrosis lesion. Tissue from CPT lesion was excised, and histological evaluation and tissue culture were performed. Histologic analysis of the soft CPT lesion showed the presence of highly cellular fibrous tissue, vascularization, and abundant extracellular matrix. Fusiform cells of mesenchymal appearance were observed but osteoblasts, osteoclasts, chondrocytes, and adipose cells were not found. There was no evidence of osteogenesis. CPT tissue cultured as explants showed, after one month of culture, evidence of osteogenesis, chondrogenesis, and adipogenesis. Cells isolated from explants of CPT tissue showed a fibroblast-like morphology and expressed the mesenchymal stromal cell (MSC) markers: CD105, CD73, and CD90 (CPT-MSC). Functional analysis showed that CPT-MSC differentiate, in vitro, into osteogenic, chondrogenic, and adipocytic cells. CPT-MSC expressed osteocalcin and agrecan. CPT-MSC produced collagen in the presence of ascorbic acid. MSC from BM of normal individuals were used as control. In summary, our results indicate that CPT tissue contains MSC with osteogenic capacity of differentiation. It is possible that CPT microenvironment may contribute to impair the osteogenic capacity of differentiation of CPT-MSC.

Amyloid-aß Peptide in olfactory mucosa and mesenchymal stromal cells of mild cognitive impairment and Alzheimer's disease patients.

Patients with mild cognitive impairment (MCI) or Alzheimer's disease (AD) might develop olfactory dysfunction that correlates with progression of disease. Alteration of olfactory neuroepithelium associated with MCI may be useful as predictor of cognitive decline. Biomarkers with higher sensitivity and specificity would allow to understand the biological progression of the pathology in association with the clinical course of the disease. In this study, magnetic resonance images, apolipoprotein E (ApoE) load, Olfactory Connecticut test and Montreal Cognitive Assessment (MoCA) indices were obtained from noncognitive impaired (NCI), MCI and AD patients. We established a culture of patient-derived olfactory stromal cells from biopsies of olfactory mucosa (OM) to test whether biological properties of mesenchymal stromal cells (MSC) are concurrent with MCI and AD psychophysical pathology. We determined the expression of amyloid Aß peptides in the neuroepithelium of tissue sections from MCI and AD, as well as in cultured cells of OM. Reduced migration and proliferation of stromal (CD90(+) ) cells in MCI and AD with respect to NCI patients was determined. A higher proportion of anosmic MCI and AD cases were concurrent with the ApoE ε4 allele. In summary, dysmetabolism of amyloid was concurrent with migration and proliferation impairment of patient-derived stem cells.

Human olfactory mucosa multipotent mesenchymal stromal cells promote survival, proliferation, and differentiation of human hematopoietic cells.

Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors and CD34(+) cells were found, at 43 days of co-culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines.