An engineered periosteum for efficient delivery of rhBMP-2 and mesenchymal progenitor cells during bone regeneration.

During bone regeneration, the periosteum acts as a carrier for key regenerative cues, delivering osteochondroprogenitor cells and crucial growth factors to the injured bone. We developed a biocompatible, 3D polycaprolactone (PCL) melt electro-written membrane to act as a mimetic periosteum. Poly (ethyl acrylate) coating of the PCL membrane allowed functionalization, mediated by fibronectin and low dose recombinant human BMP-2 (rhBMP-2) (10-25 µg/ml), resulting in efficient, sustained osteoinduction in vitro. In vivo, rhBMP-2 functionalized mimetic periosteum demonstrated regenerative potential in the treatment of rat critical-size femoral defects with highly efficient healing and functional recovery (80%-93%). Mimetic periosteum has also proven to be efficient for cell delivery, as observed through the migration of transplanted periosteum-derived mesenchymal cells to the bone defect and their survival. Ultimately, mimetic periosteum demonstrated its ability to deliver key stem cells and morphogens to an injured site, exposing a therapeutic and translational potential in vivo when combined with unprecedentedly low rhBMP-2 doses.

Non-psychoactive Cannabidiol Prevents Osteoporosis in an Animal Model and Increases Cell Viability, Proliferation, and Osteogenic Gene Expression in Human Skeletal Stem and Progenitor Cells.

Cannabidiol (CBD), the non-psychoactive component of the Cannabis sativa plant, is marketed as a potential therapeutic agent and has been studied for its roles in reducing inflammation and managing neuropathic pain. Some studies have reported that CB1 and CB2 receptor activation can attenuate and reverse bone loss in experimental animal models. Despite this, little is known about the impact of CBD on fracture healing. We investigated the effects of CBD in vitro using human osteoprogenitor cells and in vivo via murine femur fracture and osteoporosis models. In vitro mesenchymal stem cells were treated with increasing concentrations of crystalized pharmaceutical grade CBD or vehicle solution. Cell viability and proliferation were significantly increased in cells treated with CBD compared to vehicle control. Osteocalcin expression was also significantly higher in the CBD-treated human stem cells compared to vehicle control. In vivo the effect of CBD on bone mineral density and fracture healing in mice was examined using a two-phase experimental approach. Fluoxetine was used for pharmacologic induction of osteoporosis and surgical oophorectomy (OVX) was used for hormonal induction of osteoporosis. X-ray and microCT analysis showed that CBD prevented both fluoxetine- and OVX-induced osteoporosis. We found that while OVX resulted in delayed bone healing in control mice, CBD-pretreated mice exhibited normal bone healing. Collectively these in vitro and in vivo findings suggest that CBD exerts cell-specific effects which can be exploited to enhance bone metabolism. These findings also indicate that CBD usage in an osteoporotic population may positively impact bone morphology, warranting further research.

Novel hybrid biocomposites for tendon grafts: The addition of silk to polydioxanone and poly(lactide-co-caprolactone) enhances material properties, in vitro and in vivo biocompatibility.

Biopolymers play a critical role as scaffolds used in tendon and ligament (TL) regeneration. Although advanced biopolymer materials have been proposed with optimised mechanical properties, biocompatibility, degradation, and processability, it is still challenging to find the right balance between these properties. Here, we aim to develop novel hybrid biocomposites based on poly(p-dioxanone) (PDO), poly(lactide-co-caprolactone) (LCL) and silk to produce high-performance grafts suitable for TL tissue repair. Biocomposites containing 1-15% of silk were studied through a range of characterisation techniques. We then explored biocompatibility through in vitro and in vivo studies using a mouse model. We found that adding up to 5% silk increases the tensile properties, degradation rate and miscibility between PDO and LCL phases without agglomeration of silk inside the composites. Furthermore, addition of silk increases surface roughness and hydrophilicity. In vitro experiments show that the silk improved attachment of tendon-derived stem cells and proliferation over 72 h, while in vivo studies indicate that the silk can reduce the expression of pro-inflammatory cytokines after six weeks of implantation. Finally, we selected a promising biocomposite and created a prototype TL graft based on extruded fibres. We found that the tensile properties of both individual fibres and braided grafts could be suitable for anterior cruciate ligament (ACL) repair applications.

3D printed bioresorbable scaffolds for articular cartilage tissue engineering: a comparative study between neat polycaprolactone (PCL) and poly(lactide-b-ethylene glycol) (PLA-PEG) block copolymer.

This work identifies and describes different material-scaffold geometry combinations for cartilage tissue engineering (CTE). Previously reported potentially interesting scaffold geometries were tuned and printed using bioresorbable polycaprolactone and poly(lactide-b-ethylene) block copolymer. Medical grades of both polymers were 3D printed with fused filament fabrication technology within an ISO 7 classified cleanroom. Resulting scaffolds were then optically, mechanically and biologically tested. Results indicated that a few material-scaffold geometry combinations present potential for excellent cell viability as well as for an enhance of the chondrogenic properties of the cells, hence suggesting their suitability for CTE applications.

Molecular and Cellular Mechanisms of Delayed Fracture Healing in Mmp10 (Stromelysin 2) Knockout Mice.

The remodeling of the extracellular matrix is a central function in endochondral ossification and bone homeostasis. During secondary fracture healing, vascular invasion and bone growth requires the removal of the cartilage intermediate and the coordinate action of the collagenase matrix metalloproteinase (MMP)-13, produced by hypertrophic chondrocytes, and the gelatinase MMP-9, produced by cells of hematopoietic lineage. Interfering with these MMP activities results in impaired fracture healing characterized by cartilage accumulation and delayed vascularization. MMP-10, Stromelysin 2, a matrix metalloproteinase with high homology to MMP-3 (Stromelysin 1), presents a wide range of putative substrates identified in vitro, but its targets and functions in vivo and especially during fracture healing and bone homeostasis are not well defined. Here, we investigated the role of MMP-10 through bone regeneration in C57BL/6 mice. During secondary fracture healing, MMP-10 is expressed by hematopoietic cells and its maximum expression peak is associated with cartilage resorption at 14 days post fracture (dpf). In accordance with this expression pattern, when Mmp10 is globally silenced, we observed an impaired fracture-healing phenotype at 14 dpf, characterized by delayed cartilage resorption and TRAP-positive cell accumulation. This phenotype can be rescued by a non-competitive transplant of wild-type bone marrow, indicating that MMP-10 functions are required only in cells of hematopoietic linage. In addition, we found that this phenotype is a consequence of reduced gelatinase activity and the lack of proMMP-9 processing in macrophages. Our data provide evidence of the in vivo function of MMP-10 during endochondral ossification and defines the macrophages as the lead cell population in cartilage removal and vascular invasion. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Anisotropic Cryostructured Collagen Scaffolds for Efficient Delivery of RhBMP-2 and Enhanced Bone Regeneration.

In the treatment of bone non-unions, an alternative to bone autografts is the use of bone morphogenetic proteins (BMPs), e.g., BMP-2, BMP-7, with powerful osteoinductive and osteogenic properties. In clinical settings, these osteogenic factors are applied using absorbable collagen sponges for local controlled delivery. Major side effects of this strategy are derived from the supraphysiological doses of BMPs needed, which may induce ectopic bone formation, chronic inflammation, and excessive bone resorption. In order to increase the efficiency of the delivered BMPs, we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic) or trabecular bone (random distributed porosity, isotropic). We hypothesize that an anisotropic structure would enhance the osteoconductive properties of the scaffolds by increasing the regenerative performance of the provided rhBMP-2. In vitro, both scaffolds presented similar mechanical properties, rhBMP-2 retention and delivery capacity, as well as scaffold degradation time. In vivo, anisotropic scaffolds demonstrated better bone regeneration capabilities in a rat femoral critical-size defect model by increasing the defect bridging. In conclusion, anisotropic cryostructured collagen scaffolds improve bone regeneration by increasing the efficiency of rhBMP-2 mediated bone healing.

Age-related inflammation triggers skeletal stem/progenitor cell dysfunction.

Aging is associated with impaired tissue regeneration. Stem cell number and function have been identified as potential culprits. We first demonstrate a direct correlation between stem cell number and time to bone fracture union in a human patient cohort. We then devised an animal model recapitulating this age-associated decline in bone healing and identified increased cellular senescence caused by a systemic and local proinflammatory environment as the major contributor to the decline in skeletal stem/progenitor cell (SSPC) number and function. Decoupling age-associated systemic inflammation from chronological aging by using transgenic Nfkb1KO mice, we determined that the elevated inflammatory environment, and not chronological age, was responsible for the decrease in SSPC number and function. By using a pharmacological approach inhibiting NF-κB activation, we demonstrate a functional rejuvenation of aged SSPCs with decreased senescence, increased SSPC number, and increased osteogenic function. Unbiased, whole-genome RNA sequencing confirmed the reversal of the aging phenotype. Finally, in an ectopic model of bone healing, we demonstrate a functional restoration of regenerative potential in aged SSPCs. These data identify aging-associated inflammation as the cause of SSPC dysfunction and provide mechanistic insights into its reversal.

Periosteum-derived mesenchymal progenitor cells in engineered implants promote fracture healing in a critical-size defect rat model.

An attractive alternative to bone autografts is the use of autologous mesenchymal progenitor cells (MSCs) in combination with biomaterials. We compared the therapeutic potential of different sources of mesenchymal stem cells in combination with biomaterials in a bone nonunion model. A critical-size defect was created in Sprague-Dawley rats. Animals were divided into six groups, depending on the treatment to be applied: bone defect was left empty (CTL); treated with live bone allograft (LBA); hrBMP-2 in collagen scaffold (CSBMP2 ); acellular polycaprolactone scaffold (PCL group); PCL scaffold containing periosteum-derived MSCs (PCLPMSCs ) and PCL containing bone marrow-derived MSCs (PCLBMSCs ). To facilitate cell tracking, both MSCs and bone graft were isolated from green fluorescent protein (GFP)-transgenic rats. CTL group did not show any signs of healing during the radiological follow-up (n = 6). In the LBA group, all the animals showed bone bridging (n = 6) whereas in the CSBMP2 group, four out of six animals demonstrated healing. In PCL and PCLPMSCs groups, a reduced number of animals showed radiological healing, whereas no healing was detected in the PCLBMSCs group. Using microcomputed tomography, the bone volume filling the defect was quantified, showing significant new bone formation in the LBA, CSBMP2 , and PCLPMSCs groups when compared with the CTL group. At 10 weeks, GFP positive cells were detected only in the LBA group and restricted to the outer cortical bone in close contact with the periosteum. Tracking of cellular implants demonstrated significant survival of the PMSCs when compared with BMSCs. In conclusion, PMSCs improve bone regeneration being suitable for mimetic autograft design.

Tissue Mimicry in Morphology and Composition Promotes Hierarchical Matrix Remodeling of Invading Stem Cells in Osteochondral and Meniscus Scaffolds.

An integral approach toward in situ tissue engineering through scaffolds that mimic tissue with regard to both tissue architecture and biochemical composition is presented. Monolithic osteochondral and meniscus scaffolds are prepared with tissue analog layered biochemical composition and perpendicularly oriented continuous micropores by a newly developed cryostructuring technology. These scaffolds enable rapid cell ingrowth and induce zonal-specific matrix synthesis of human multipotent mesenchymal stromal cells solely through their design without the need for supplementation of soluble factors such as growth factors.

Autologous bioscaffolds based on different concentrations of platelet rich plasma and synovial fluid as a vehicle for mesenchymal stem cells.

In the field of tissue engineering, diverse types of bioscaffolds are being developed currently for osteochondral defect applications. In this work, a novel scaffold based on platelet rich plasma (PRP) and hyaluronic acid with mesenchymal stem cells (MSCs) has been evaluated to observe its effect on immobilized cells. The bioscaffolds were prepared by mixing different volumes of synovial fluid (SF) with PRP from patients obtaining three formulations at PRP-SF ratios of 3:1, 1:1 and 1:3 (v/v). The live/dead staining revealed that although the cell number of each type of bioscaffold was different, these this constructs provide cells with a suitable environment for their viability and proliferation. Moreover, immobilized MSCs showed their ability to secrete fibrinolytic enzymes, which vary depending on the fibrin amount of the scaffold. Immunohistochemical analysis revealed the positive staining for collagen type II in all cases, proving the biologic action of SF derived MSCs together with the suitable characteristics of the bioscaffold for chondrogenic differentiation. Considering all these aspects, this study demonstrates that these cells-based constructs represent an attractive method for cell immobilization, achieving completely autologous and biocompatible scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 377-385, 2018.

* Human Amniotic Mesenchymal Stromal Cells as Favorable Source for Cartilage Repair.

INTRODUCTION: Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. MATERIAL AND METHODS: A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. RESULTS: HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. CONCLUSION: HAM is a useful material for articular cartilage repair in vitro when used as scaffold. In combination with hAMSCs, HAM showed better potential for cartilage repair with similar reparation capacity than chondrocytes.

Modulation of Synovial Fluid-Derived Mesenchymal Stem Cells by Intra-Articular and Intraosseous Platelet Rich Plasma Administration.

The aim of this study was to evaluate the effect of intra-articular (IA) or a combination of intra-articular and intraosseous (IO) infiltration of Platelet Rich Plasma (PRP) on the cellular content of synovial fluid (SF) of osteoarthritic patients. Thirty-one patients received a single infiltration of PRP either in the IA space (n = 14) or in the IA space together with two IO infiltrations, one in the medial femoral condyle and one in the tibial plateau (n = 17). SF was collected before and after one week of the infiltration. The presence in the SF of mesenchymal stem cells (MSCs), monocytes, and lymphocytes was determined and quantified by flow cytometry. The number and identity of the MSCs were further confirmed by colony-forming and differentiation assays. PRP infiltration into the subchondral bone (SB) and the IA space induced a reduction in the population of MSCs in the SF. This reduction in MSCs was further confirmed by colony-forming (CFU-F) assay. On the contrary, IA infiltration alone did not cause variations in any of the cellular populations by flow cytometry or CFU-F assay. The SF of osteoarthritic patients contains a population of MSCs that can be modulated by PRP infiltration of the SB compartment.

Combination of Intra-Articular and Intraosseous Injections of Platelet Rich Plasma for Severe Knee Osteoarthritis: A Pilot Study.

The aim of this study was to assess a novel approach to treating severe knee osteoarthritis by targeting synovial membrane, superficial articular cartilage, synovial fluid, and subchondral bone by combining intra-articular injections and intraosseous infiltrations of platelet rich plasma. We explored a new strategy consisting of intraosseous infiltrations of platelet rich plasma into the subchondral bone in combination with the conventional intra-articular injection in order to tackle several knee joint tissues simultaneously. We assessed the clinical outcomes through osteoarthritis outcome score (KOOS) and the inflammatory response by quantifying mesenchymal stem cells in synovial fluid. There was a significant pain reduction in the KOOS from baseline (61.55 ± 14.11) to week 24 (74.60 ± 19.19), after treatment (p = 0.008), in the secondary outcomes (symptoms, p = 0.004; ADL, p = 0.022; sport/rec., p = 0.017; QOL, p = 0.012), as well as VAS score (p < 0.001) and Lequesne Index (p = 0.008). The presence of mesenchymal stem cells in synovial fluid and colony-forming cells one week after treatment decreased substantially from 7.98 ± 8.21 MSC/µL to 4.04 ± 5.36 MSC/µL (p = 0.019) and from 601.75 ± 312.30 to 139.19 ± 123.61 (p = 0.012), respectively. Intra-articular injections combined with intraosseous infiltrations of platelet rich plasma reduce pain and mesenchymal stem cells in synovial fluid, besides significantly improving knee joint function in patients with severe knee osteoarthritis. This trial is registered on EudraCT with the number 2013-003982-32.

Hypoxia and Reactive Oxygen Species Homeostasis in Mesenchymal Progenitor Cells Define a Molecular Mechanism for Fracture Nonunion.

Fracture nonunion is a major complication of bone fracture regeneration and repair. The molecular mechanisms that result in fracture nonunion appearance are not fully determined. We hypothesized that fracture nonunion results from the failure of hypoxia and hematoma, the primary signals in response to bone injury, to trigger Bmp2 expression by mesenchymal progenitor cells (MSCs). Using a model of nonstabilized fracture healing in transgenic 5'Bmp2BAC mice we determined that Bmp2 expression appears in close association with hypoxic tissue and hematoma during the early phases of fracture healing. In addition, BMP2 expression is induced when human periosteum explants are exposed to hypoxia ex vivo. Transient interference of hypoxia signaling in vivo with PX-12, a thioredoxin inhibitor, results in reduced Bmp2 expression, impaired fracture callus formation and atrophic-like nonunion by a HIF-1α independent mechanism. In isolated human periosteum-derived MSCs, BMP2 expression could be induced with the addition of platelets concentrate lysate but not with hypoxia treatment, confirming HIF-1α-independent BMP2 expression. Interestingly, in isolated human periosteum-derived mesenchymal progenitor cells, inhibition of BMP2 expression by PX-12 is accomplished only under hypoxic conditions seemingly through dis-regulation of reactive oxygen species (ROS) levels. In conclusion, we provide evidence of a molecular mechanism of hypoxia-dependent BMP2 expression in MSCs where interference with ROS homeostasis specifies fracture nonunion-like appearance in vivo through inhibition of Bmp2 expression. Stem Cells 2016;34:2342-2353.

Alternative protocols to induce chondrogenic differentiation: transforming growth factor-ß superfamily.

Mesenchymal stem cells (MSCs) are an accepted candidate for cell-based therapy of multiple diseases. The interest in MSCs and their possible application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently, like aggregation and transforming growth factor beta (TGFß) delivery, hypoxia has been indicated as crucial for complete chondrogenesis. The aim of this study was to test different culture conditions for directing stem cell differentiation into the chondrogenic lineage in vitro by testing different TGFß superfamily members into the culture media under normoxic conditions. All chondrogenic culture conditions used allowed the differentiation of bone marrow-MSCs (BM-MSCs) into chondrogenic lineage. Chondrogenic induction capacity depended on the growth factor added to the culture media. In particular, the chondrogenic culture condition that better induced chondrogenesis was the medium that included the combination of three growth factors: bone morphogenetic protein-2 (BMP-2), BMP-7 and TGFß-3. In this culture media, differentiated cells showed the highest levels expression of two markers of chondrogenesis, SOX9 and COL2A1, compared to the control points (p < 0.05, two-tailed t test). In our experimental conditions, the combination of BMP-2, BMP-7 and TGFß-3 was the most effective in promoting chondrogenesis of BM-MSCs. These results underline the importance of determining in each experimental design the best protocol for in vitro directing stem cell differentiation into the chondrogenic lineage.

Effects of severe hypoxia on bone marrow mesenchymal stem cells differentiation potential.

Background. The interests in mesenchymal stem cells (MSCs) and their application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently hypoxia has been indicated as crucial for complete chondrogenesis. We aimed at analyzing bone marrow MSCs (BM-MSCs) differentiation capacity under normoxic and severe hypoxic culture conditions. Methods. MSCs were characterized by flow cytometry and differentiated towards adipocytes, osteoblasts, and chondrocytes under normoxic or severe hypoxic conditions. The differentiations were confirmed comparing each treated point with a control point made of cells grown in DMEM and fetal bovine serum (FBS). Results. BM-MSCs from the donors displayed only few phenotypical differences in surface antigens expressions. Analyzing marker genes expression levels of the treated cells compared to their control point for each lineage showed a good differentiation in normoxic conditions and the absence of this differentiation capacity in severe hypoxic cultures. Conclusions. In our experimental conditions, severe hypoxia affects the in vitro differentiation potential of BM-MSCs. Adipogenic, osteogenic, and chondrogenic differentiations are absent in severe hypoxic conditions. Our work underlines that severe hypoxia slows cell differentiation by means of molecular mechanisms since a decrease in the expression of adipocyte-, osteoblast-, and chondrocyte-specific genes was observed.

Characterization of microRNA expression profiles in normal and osteoarthritic human chondrocytes.

BACKGROUND: Osteoarthritis (OA) is a multifactorial disease characterized by destruction of the articular cartilage due to environmental, mechanical and genetic components. The genetics of OA is complex and is not completely understood. Recent works have demonstrated the importance of microRNAs (miRNAs) in cartilage function. MiRNAs are a class of small noncoding RNAs that regulate gene expression and are involved in different cellular process: apoptosis, proliferation, development, glucose and lipid metabolism. The aim of this study was to identify and characterize the expression profile of miRNAs in normal and OA chondrocytes and to determine their role in the OA. METHODS: Chondrocytes were moved to aggregate culture and evaluated using histological and qPCR techniques. miRNAs were isolated and analyzed using the Agilent Human miRNA Microarray. RESULTS: Of the 723 miRNAs analyzed, 7 miRNAs showed a statistically significant differential expression. Amongst these 7 human miRNAs, 1 was up-regulated in OA chondrocytes (hsa-miR-483-5p) and 6 were up-regulated in normal chondrocytes (hsa-miR-149*, hsa-miR-582-3p, hsa-miR-1227, hsa-miR-634, hsa-miR-576-5p and hsa-miR-641). These profiling results were validated by the detection of some selected miRNAs by qPCR. In silico analyses predicted that key molecular pathways potentially altered by the miRNAs differentially expressed in normal and OA chondrocytes include TGF-beta, Wnt, Erb and mTOR signalling; all of them implicated in the development, maintenance and destruction of articular cartilage. CONCLUSIONS: We have identified 7 miRNAs differentially expressed in OA and normal chondrocytes. Our potential miRNA target predictions and the signalling cascades altered by the differentially expressed miRNAs supports the potential involvement of the detected miRNAs in OA pathology. Due to the importance of miRNA in mediating the translation of target mRNA into protein, the identification of these miRNAs differentially expressed in normal and OA chondrocyte micropellets could have important diagnostic and therapeutic potential. Further studies are needed to know the function of these miRNAs, including the search of their target mRNA genes, which could lead to the development of novel therapeutic strategies for the OA treatment.

Cryopreservation effect on proliferative and chondrogenic potential of human chondrocytes isolated from superficial and deep cartilage.

OBJECTIVES: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. MATERIALS AND METHODOLOGY: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. RESULTS: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). CONCLUSIONS: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes.

Human amniotic membrane as an alternative source of stem cells for regenerative medicine.

The human amniotic membrane (HAM) is a highly abundant and readily available tissue. This amniotic tissue has considerable advantageous characteristics to be considered as an attractive material in the field of regenerative medicine. It has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Since it is discarded post-partum it may be useful for regenerative medicine and cell therapy. Amniotic membranes have already been used extensively as biologic dressings in ophthalmic, abdominal and plastic surgery. HAM contains two cell types, from different embryological origins, which display some characteristic properties of stem cells. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. Both populations have similar immunophenotype and multipotential for in vitro differentiation into the major mesodermal lineages, however they differ in cell yield. Therefore, HAM has been proposed as a good candidate to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues.

Bone marrow cells immunomagnetically selected for CD271+ antigen promote in vitro the repair of articular cartilage defects.

OBJECTIVE: The purposes of this project were to quantify the cells expressing the mesenchymal stem cell (MSC) marker CD271 in synovial membranes from human osteoarthritic (OA) and healthy joints, and to determine if those CD271 cells were involved in spontaneous human cartilage repair and were beneficial for the repair of human articular cartilage defects. METHODS: The coexpression of CD44/CD271, CD90/CD271, and CD105/CD271 antigens was determined by immunofluorescence in OA and healthy synovial membranes and during spontaneous cartilage repair. Isolated MSCs from the bone marrow of four OA patients (mean age: 64 years) were magnetically separated into MSC CD271+ and MSC CD271- subsets. The separated cell subsets were then implanted into 2 mm focal defects of articular cartilage. These implants were cultured in chondrogenic differentiation medium supplemented with recombinant human transforming growth factor-beta3 for 8 weeks. The repair tissues were analyzed by histochemistry (hematoxylin-eosin and safranin O) and immunohistochemistry for collage types I and II. RESULTS: Cells expressing the CD271 antigen were diffusely distributed in OA synovial membranes and localized in the subintimal zone in healthy synovial membranes. The number of cells expressing MSC markers was higher in OA synovial membranes than in synovia from healthy joints, corresponding to the highest level of coexpression of CD90/CD271 antigens (9.8% vs. 2.6%). Spontaneous repair tissue showed more cells expressing the CD271 antigen (9.9% ± 4.0%). The highest levels of expression were found to be associated with CD44; 64% of positive CD271 cells coexpressed the CD44 antigen. In both implant cell types, the repair tissue morphology resembled articular cartilage, having an extracellular matrix with a hyaline aspect and numerous lacunae containing cells, and was immunopositive for collagen types I and II. Statistical analyses of the repair tissue demonstrated that the implantation of MSC CD271+ provided such benefits as a greater filling of the chondral defect and better integration between the repair tissue and native cartilage. Safranin O staining of repair tissue was negative in implants of MSC CD271- but more positive in implants with MSC CD271+. The overall histologic score for CD271- implants was 9.5 ± 0.89 and 12.19 ± 1.01 for CD271+ implants. CONCLUSIONS: Synovial membranes from OA patients contain more cells expressing CD271 antigen than those from healthy joints, and spontaneous cartilage repair tissue contains cells positive for CD271 antigen. These data suggest the involvement of CD271 antigen in spontaneous cartilage repair and indicate that the cell subset MSC CD271+ provides higher quality chondral repair than the CD271- subset.