Posttraumatic osteoarthritis (PTOA) is mostly treated via corticosteroid administration, and total joint arthroplasty continues to be the sole effective intervention in severe conditions. To assess the therapeutic potential of CCR2 targeting in PTOA, we used biodegradable microplates (mu PLs) to achieve a slow and sustained intraarticular release of the CCR2 inhibitor RS504393 into injured knees and followed joint damage during disease progression. RS504393-loaded mu PLs (RS-mu PLs) were fabricated via a template-replica molding technique. A mixture of poly(lactic-co-glycolic acid) (PLGA) and RS504393 was deposited into 20 x 10 mu m (length x height) wells in a polyvinyl alcohol (PVA) square-patterned template. After physicochemical and toxicological characterizations, the RS504393 release profile from mu PL was assessed in PBS buffer. C57BL/6 J male mice were subjected to destabilization of the medial meniscus (DMM)/sham surgery, and RS-mu PLs (1 mg/kg) were administered intraarticularly 1 week postsurgery. Administrations were repeated at 4 and 7 weeks post-DMM. Drug free-mu PLs (DF-mu PLs) and saline injections were performed as controls. Mice were euthanized at 4 and 10 weeks post-DMM, corresponding to the early and severe PTOA stages, respectively. Knees were evaluated for cartilage structure score (ACS, H&E), matrix loss (safranin O score), osteophyte formation and maturation from cartilage to bone (cartilage quantification), and subchondral plate thickness. The RS-mu PL architecture ensured the sustained release of CCR2 inhibitors over several weeks, with similar to 20% of RS504393 still available at 21 days. This prolonged release improved cartilage structure and reduced bone damage and synovial hyperplasia at both PTOA stages. Extracellular matrix loss was also attenuated, although with less efficacy. The results indicate that local sustained delivery is needed to optimize CCR2-targeted therapies.

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.

Eradicating leukemia requires a deep understanding of the interaction between leukemic cells and their protective microenvironment. The CXCL12/CXCR4 axis has been postulated as a critical pathway dictating leukemia stem cell (LSC) chemoresistance in AML due to its role in controlling cellular egress from the marrow. Nevertheless, the cellular source of CXCL12 in the acute myeloid leukemia (AML) microenvironment and the mechanism by which CXCL12 exerts its protective role in vivo remain unresolved. Here, we show that CXCL12 produced by Prx1+ mesenchymal cells but not by mature osteolineage cells provide the necessary cues for the maintenance of LSCs in the marrow of an MLL::AF9-induced AML model. Prx1+ cells promote survival of LSCs by modulating energy metabolism and the REDOX balance in LSCs. Deletion of Cxcl12 leads to the accumulation of reactive oxygen species and DNA damage in LSCs, impairing their ability to perpetuate leukemia in transplantation experiments, a defect that can be attenuated by antioxidant therapy. Importantly, our data suggest that this phenomenon appears to be conserved in human patients. Hence, we have identified Prx1+ mesenchymal cells as an integral part of the complex niche-AML metabolic intertwining, pointing towards CXCL12/CXCR4 as a target to eradicate parenchymal LSCs in AML.

The remodelling 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. (c) 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).

Despite the bone marrow microenvironment being widely recognised as a key player in cancer research, the current animal models that represent a human haematopoietic system lack the contribution of the humanised marrow microenvironment. Here we describe a murine model that relies on the combination of an orthotopic humanised tissue-engineered bone construct (ohTEBC) with patient-specific bone marrow (BM) cells to create a humanised bone marrow (hBM) niche capable of supporting the engraftment of human haematopoietic cells. Results showed that this model supports the engraftment of human CD34+ cells from a healthy BM with human haematopoietic cells migrating into the mouse BM, human BM compartment, spleen and peripheral blood. We compared these results with the engraftment capacity of human CD34+ cells obtained from patients with multiple myeloma (MM). We demonstrated that CD34+ cells derived from a diseased BM had a reduced engraftment potential compared to healthy patients and that a higher cell dose is required to achieve engraftment of human haematopoietic cells in peripheral blood. Finally, we observed that haematopoietic cells obtained from the mobilised peripheral blood of patients yields a higher number of CD34+, overcoming this problem. In conclusion, this humanised mouse model has potential as a unique and patient-specific pre-clinical platform for the study of tumour-microenvironment interactions, including human bone and haematopoietic cells, and could, in the future, serve as a drug testing platform.

Background Mesenchymal stromal cells are a safe and promising option to treat knee osteoarthritis as previously demonstrated in different clinical trials. However, their efficacy, optimal dose and addition of adjuvants must be determined. Here, we evaluated the clinical effects of a dose of 100 x 10(6)bone marrow mesenchymal stromal cells (BM-MSCs) in combination with Platelet Rich Plasma (PRGF (R)) as adjuvant in a randomized clinical trial. Methods A phase II, multicenter, randomized clinical trial with active control was conducted. Sixty patients diagnosed with knee OA were randomly assigned to 3 weekly doses of PRGF (R) or intraarticular administration of 100 x 10(6)cultured autologous BM-MSCs plus PRGF (R). Patients were followed up for 12 months, and pain and function were assessed using VAS and WOMAC and by measuring the knee range of motion range. X-ray and magnetic resonance imaging analyses were performed to analyze joint damage. Results No adverse effects were reported after BM-MSC administration or during follow-up. According to VAS, the mean value (SD) for PRGF (R) and BM-MSC with PRGF (R) went from 5 (1.8) to 4.5 (2.2) (p = 0.389) and from 5.3 (1.9) to 3.5 (2.5) (p = 0.01), respectively at 12 months. In WOMAC, the mean (SD) baseline and 12-month overall WOMAC scores in patients treated with PRGF (R) was 31.9 (16.2) and 22.3 (15.8) respectively (p = 0.002) while that for patients treated with BM-MSC plus PRGF (R) was 33.4 (18.7) and 23.0 (16.6) (p = 0.053). Although statistical significances between groups have not been detected, only patients being treated with BM-MSC plus PRGF (R) could be considered as a OA treatment responders following OARSI criteria. X-ray and MRI (WORMS protocol) revealed no changes in knee joint space width or joint damage. Conclusions Treatment with BM-MSC associated with PRGF (R) was shown to be a viable therapeutic option for osteoarthritis of the knee, with clinical improvement at the end of follow-up. Further phase III clinical trials would be necessary to confirm the efficacy. Trial registrationClinical Trials.gov identifier NCT02365142. EudraCT No: 2011-006036-23

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.

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.

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 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. (c) 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 377-385, 2018.

Acceleration of the consolidation of the distracted bone is a relevant medical need. As a platform to improve in vivo bone engineering, we developed a novel distraction osteogenesis (DO) model in a rabbit large bone (femur) and tested if the application of cultured bone marrow stromal cells (BMSCs) immediately after the osteotomy promotes the formation of bone. This report consists of two components, an animal study to evaluate the quality of the regenerate following different treatments and an in vitro study to evaluate osteogenic potential of BMSC cultures. To illuminate the mechanism of action of injected cells, we tested stem cell cultures enriched in osteogenic-BMSCs (O-BMSCs) as compared with cultures enriched in non-osteogenic BMSCs (NO-BMSCs). Finally, we included a group of animals treated with biomaterials (fibrin and ground cortical bone) in addition to cells. Injection of O-BMSCs promoted the maturity of distracted callus and decreased fibrosis. When combined with biomaterials, O-BMSCs modified the ossification pattern from endochondral to intramembranous type. The use of NO-BMSCs not only did not increase the maturity but also increased porosity of the bone. These preclinical results indicate that the BMSC cultures must be tested in vitro prior to clinical use, since a number of factors may influence their outcome in bone formation. We hypothesize that the use of osteogenic BMSCs and biomaterials could be clinically beneficial to shorten the consolidation period of the distraction and the total period of bone lengthening. (C) 2018 Elsevier Ltd. All rights reserved.