The aim of the study was to determine the long-term effect of transplantation of adipose-derived stromal cells (ADSCs) in a preclinical model of ischemia/reperfusion (I/R). I/R was induced in 28 Goettingen minipigs by 120 min of coronary artery occlusion followed by reperfusion. Nine days later, surviving animals were allocated to receive transendocardial injection of a mean of 213.6 ± 41.78 million green fluorescent protein (GFP)-expressing ADSCs (n = 7) or culture medium as control (n = 9). Heart function, cell engraftment, and histological analysis were performed 3 months after transplantation. Transplantation of ADSCs induced a statistically significant long-lasting (3 months) improvement in cardiac function and geometry in comparison with control animals. Functional improvement was associated with an increase in angiogenesis and vasculogenesis and a positive effect on heart remodeling with a decrease in fibrosis and cardiac hypertrophy in animals treated with ADSCs. Despite the lack of cell engraftment after 3 months, ADSC transplantation induced changes in the ratio between MMP/TIMP. Our results indicate that transplantation of ADSCs, despite the lack of long-term significant cell engraftment, increases vessel density and prevents adverse remodeling in a clinically relevant model of myocardial infarction, strongly suggesting a paracrine-mediated effect. ADSCs thus constitute an attractive candidate for the treatment of myocardial infarction.

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 0 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-1a 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-1a-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 SIGNIFICANCE STATEMENTFracture nonunion is a major cause of chronic pain and disability. However, the molecular mechanisms that result in impaired fracture healing are not yet determined. The regenerative capability of bone tissue resides in the mesenchymal progenitor cells (MSCs) that can differentiate into chondrocytes and osteoblasts in response to several stimuli derived of bone injury. Here we demonstrate that fracture-healing initiation is highly dependent on the management by MSCs of hypoxiaderived intracellular oxidative stress. This study identifies a mechanism for fracture nonunion appearance that may allow the prevention and non-surgical treatment of fracture nonunions.

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 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.

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 PCL (polycaprolactone) and PLA PEG- (poly(lactide-b-ethylene) block copolymer). Medical grades of both polymers were 3D printed with FFF 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.

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).

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