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.
BackgroundTGF-β has been described as a mediator of fibrosis and scarring. Several studies achieved reduction in experimental scarring through the inhibition of TGF-β. Fibroblasts have been defined as the cell population originating fibrosis, blocking fibroblast invasion may impair epidural fibrosis appearance. For this purpose, biocompatible materials used as mechanical barriers and a TGF-β inhibitor peptide were evaluated in the reduction of epidural fibrosis.MethodsA L6 laminectomy was performed in 40 New Zealand white rabbits. Divided into four groups, each rabbit was assigned to receive either collagen sponge scaffold (CS group), gelatin-based gel (GCP group), P144® (iTGFβ group), or left untreated (control group). Four weeks after surgery, cell density, collagen content, and new bone formation of the scar area were determined by histomorphometry. Two experienced pathologists scored dura mater adhesion, scar density, and inflammatory infiltrate in a blinded manner.ResultsIn all groups, laminectomy site was filled with fibrous tissue and the dura mater presented adhesions. Only GCP group presented a significant reduction in collagen content and scar density.ConclusionGCP treatment reduces epidural fibrosis although did not prevent dura mater adhesion completely.Electronic supplementary materialThe online version of this article (10.1186/s13018-018-0781-6) contains supplementary material, which is available to authorized users.
Achilles tendon rupture is a frequent injury with an increasing incidence. After clinical surgical repair, aimed at suturing the tendon stumps back into their original position, the repaired Achilles tendon is often plastically deformed and mechanically less strong than the pre-injured tissue, with muscle fatty degeneration contributing to function loss. Despite clinical outcomes, pre-clinical research has mainly focused on tendon structural repair, with a lack of knowledge regarding injury progression from tendon to muscle and its consequences on muscle degenerative/regenerative processes and function. Here, we characterize the morphological changes in the tendon, the myotendinous junction and muscle belly in a mouse model of Achilles tendon complete rupture, finding cellular and fatty infiltration, fibrotic tissue accumulation, muscle stem cell decline and collagen fiber disorganization. We use novel imaging technologies to accurately relate structural alterations in tendon fibers to pathological changes, which further explain the loss of muscle mechanical function after tendon rupture. The treatment of tendon injuries remains a challenge for orthopedics. Thus, the main goal of this study is to bridge the gap between clinicians’ knowledge and research to address the underlying pathophysiology of ruptured Achilles tendon and its consequences in the gastrocnemius. Such studies are necessary if current practices in regenerative medicine for Achilles tendon ruptures are to be improved.
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