RESUMEN
Three different heterologous substitutes for bone regeneration, manufactured with equine-derived cortical powder (CP), cancellous chips (CC) and demineralized bone matrix granules (DBM), were compared in in vitro and in vivo settings. We tested: a commercially available bone paste (Osteoplant-Activagen™, consisting of aqueous collagenous carrier, CP, DBM; named A); a second-generation injectable paste (20 kDa polyethylene glycol/hydroxypropyl-methyl cellulose-based hydrogel, CP, DBM; B); a pre-formed bone filler (400 kDa polyethylene oxide/hydroxypropyl-methyl cellulose-based hydrogel, CP, CC, DBM; C). Vitamin C acted as a visco-modulator during C and B ß-rays sterilization, modifying graft injectability. For each filler, we examined dissolution in culture medium, gene expression of the substitute-exposed osteogenically-induced human bone marrow stromal cells (hBMSC), and performance in a rabbit bone defect model. A dissolved after 1 h, while fragmentation of B peaked after 8 h. C remained unaltered for 2 days, but affected the microenvironmental pH, slowing the proliferation of exposed cells. B-exposed hBMSC overexpressed bone sialoprotein, osteocalcin and RUNX2. For all fillers histological results evidenced bridged lesion margins, marrow replenishment and bone-remodeling. However, B-treated lesions displayed a metachromatic type II collagen-rich matrix with prehypertrophic-like cells, matching the in vitro expression of cartilage-specific markers, and suggesting a possible application of B/C double-layer monolithic osteochondral plugs for full-thickness articular defects.
Asunto(s)
Materiales Biocompatibles/química , Sustitutos de Huesos/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Animales , Huesos/lesiones , Línea Celular , Humanos , Células Madre Mesenquimatosas , Conejos , ReologíaRESUMEN
The clinical need for improved human autologous chondrocyte transplantation has motivated the use of different biomaterials, which are aimed at fixing the cells in the defect area and permit their proliferation and differentiation. The maintenance of the original phenotype by isolated chondrocytes grown in vitro is an important requisite for their use in repairing damaged articular cartilage. The methods to verify the expression of cartilage-specific molecules usually involve destructive procedures to recover the cells from the scaffolds for tests. The aim of our study was to find a soluble marker able to attest the occurrence of a differentiation process by chondrocytes grown onto a biomaterial used for cell transplantation. We turned our attention to cathepsin B which is known to be abnormally synthesized in de-differentiated chondrocytes and scarcely produced in the differentiated ones. The production of cathepsin B by human articular chondrocytes expanded in vitro and then grown onto a hyaluronan-based polymer derivative (Hyaff-11) three-dimensional scaffold was evaluated with a specific enzyme-immunoassay at different experimental times together with the expression of mRNA by real-time PCR. We showed that cathepsin B, which is abundantly produced by chondrocytes grown in a monolayer culture, decreases significantly after the cells are seeded onto the scaffold, giving further evidence of a re-differentiation process. This result suggests cathepsin B a practical soluble marker to evaluate the "good" quality of transplantable constructs.
Asunto(s)
Cartílago Articular/trasplante , Catepsina B/metabolismo , Ingeniería de Tejidos/métodos , Materiales Biocompatibles , Biomarcadores , Fenómenos Biomecánicos , Catepsina B/genética , Diferenciación Celular , División Celular , Condrocitos/citología , Condrocitos/metabolismo , Condrocitos/trasplante , Expresión Génica , Humanos , Ácido Hialurónico/análogos & derivados , Inmunohistoquímica , Ensayo de Materiales , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solubilidad , Trasplante AutólogoRESUMEN
Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. Such scaffolds should provide a preformed three-dimensional shape and prevent cells from escaping into the articular cavity. Furthermore, these constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and their phenotype redifferentiation. The aim of this study was to verify the ability of HYAFF 11, a recently developed hyaluronic-acid-based biodegradable polymer, to support the growth of human chondrocytes and to maintain their original phenotype. This capability was assessed by the evaluation of collagen types I, II and aggrecan mRNA expression. Immunohistochemical analyses were also performed to evaluate collagen types I, II and proteoglycans synthesis. A field emission in lens scanning microscopy was utilized to verify the interactions between the cells and the biomaterial. Our data indicate that human chondrocytes seeded on HYAFF 11 express and produce collagen type II and aggrecan and downregulate the production of collagen type I. These results provide an in vitro demonstration for the therapeutic potential of HYAFF 11 as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects.
Asunto(s)
Materiales Biocompatibles , Condrocitos/citología , Proteínas de la Matriz Extracelular , Ácido Hialurónico/análogos & derivados , Agrecanos , Diferenciación Celular , División Celular , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/ultraestructura , Colágeno Tipo I/genética , Colágeno Tipo II/genética , Humanos , Inmunohistoquímica , Lectinas Tipo C , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Proteoglicanos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In reconstructive surgery, implantable devices are used to supply a missing function. In tissue engineering, biomaterials serve to guide and eventually deliver cells and/or molecules where a tissue regenerative response is needed. The host organism always reacts to implants of any biomaterial, in some instances even triggering a local cascade of events called the foreign body response (FBR), whose mechanisms are well defined. What has yet to be completely unraveled are the biomarkers systemically mirroring the FBR and the regeneration processes, which would be helpful for assessing the therapeutic efficacy of the bioscaffold. Our goal was to identify a biomarker fingerprint of the systemic reaction of host response to bioscaffold implants. Different biomaterials chosen for their osteoconductive properties, including collagen, hydroxyapatite, in foam or granules, and poly-ε-caprolactone, were implanted in immunocompetent mice. We analyzed serum concentrations of cells and cytokines involved in the inflammatory/immune response, and the histological features of grafts. Within two weeks after implantation, a wave of proinflammatory cytokines was flowing in the blood stream and the concentration of blood cells changed, revealing specific patterns depending on the chemistry and structure of the implanted biomaterials. Cells secreting pro-inflammatory, chemoactractant, and pro-angiogenic cytokines required for the early events in tissue repair were locally recruited because of the presence of a bioscaffold.