Many substances are used in the production of biomaterials: metals (titanium), ceramics (alumina), synthetic polymers (polyurethanes, silicones, polyglycolic acid (PGA), polylactic acid (PLA), copolymers of lactic and glycolic acids (PLGA), polyanhydrides, polyorthoesters) and natural polymers (chitosan, glycosaminoglycans, collagen). With the rapid development in tissue engineering, these different biomaterials have been used as three-dimensional scaffolds and cell transplant devices. The principal biochemical and biological characteristics of the collagen-based biomaterials are presented, including their interactions with cells (fibroblasts), distinct from those of synthetic polymers, and their potential use in gene therapy through the formation of neo-organs or organoids.

Collagen-based films and sponges are widely used as biomaterials. The rate of their biodegradation can be reduced by treating them with different cross-linking agents. The efficiency of different reticulation procedures on thermal stability (measured by differential scanning calorimetry) and susceptibility to bacterial collagenase digestion of the final material (films or sponges) is compared. The chemical agents used on collagen gels or directly on collagen sponges and films were glutaraldehyde (GTA), hexamethylene diisocyanate (HMDC), cyanamide, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and the two acyl azide methods (with hydrazine or diphenylphosphorylazide (DPPA)) developed in the authors' laboratory. Under these experimental conditions, collagen stabilization by the different agents increased in the following order: cyanamide < EDC < HMDC < hydrazine < DPPA < GTA. Sponges with the highest thermal stability were obtained by treating collagen gel with 0.5% GTA for 1 h at pH 5.5 (initial T~=75.8°C; after 3 months, Ta=75.6°C). Direct treatment of collagen films with DPPA gave similar thermal stability (Ta=72.6 °C) and collagenase resistance when compared with treatment with 0.6% GTA for 96 h (Td =74.6 °C). It is demonstrated that collagen sponges and films can be prepared with a wide range of thermal stability (49-75.8 °C) and collagenase digestion resistance (10-100%). Recent biocompatibility studies show, however, that DPPA and EDC are the best choices for preparing cross-linked collagen sponges and films.

We investigated the effects of bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-␤ superfamily, on the regulation of the chondrocyte phenotype, and we identified signaling molecules involved in this regulation. BMP-2 triggers three concomitant responses in mouse primary chondrocytes and chondrocytic MC615 cells. First, BMP-2 stimulates expression or synthesis of type II collagen. Second, BMP-2 induces expression of molecular markers characteristic of pre-and hypertrophic chondrocytes, such as Indian hedgehog, parathyroid hormone/parathyroid hormonerelated peptide receptor, type X collagen, and alkaline phosphatase. Third, BMP-2 induces osteocalcin expression, a specific trait of osteoblasts. Constitutively active forms of transforming growth factor-␤ family type I receptors and Smad proteins were overexpressed to address their role in this process. Activin receptor-like kinase (ALK)-1, ALK-2, ALK-3, and ALK-6 were able to reproduce the hypertrophic maturation of chondrocytes induced by BMP-2. In addition, ALK-2 mimicked further the osteoblastic differentiation of chondrocytes induced by BMP-2. In the presence of BMP-2, Smad1, Smad5, and Smad8 potentiated the hypertrophic maturation of chondrocytes, but failed to induce osteocalcin expression. Smad6 and Smad7 impaired chondrocytic expression and osteoblastic differentiation induced by BMP-2. Thus, our results indicate that Smad-mediated pathways are essential for the regulation of the different steps of chondrocyte and osteoblast differentiation and suggest that additional Smad-independent pathways might be activated by ALK-2.The development of long bones involves first the formation of cartilage primordia, which prefigure the future skeletal elements, and the replacement of the cartilage by bone. The formation of cartilage is initiated by the condensation and differentiation of mesenchymal cells into chondrocytes. This commitment to the skeletal lineage progresses through a switch in gene activation and results in the production of a cartilage matrix, which contains predominantly type II collagen and the proteoglycan aggrecan. During replacement of cartilage by bone through the sequence of events called endochondral ossification, chondrocytes enter a process of maturation, characterized by cellular hypertrophy and onset of type X collagen expression (reviewed in Ref. 1). This is followed by vascular invasion, matrix degradation, and replacement of the cartilage by bone marrow and trabecular bone.Chondrocyte differentiation and maturation is thus a central cellular aspect of skeletal development, and a molecular understanding of skeletogenesis requires an understanding of how expression of the chondrocytic phenotype is regulated. The fate of the skeletal cells and their precursors is controlled by the extracellular matrix, growth factors, hormones, and cytokines. Bone morphogenetic proteins (BMPs) 1 play a particularly important role in skeletal formation. Although BMPs have been shown to have a broad spectrum of action on proliferati...

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Mechanical loading is important in tissue formation and remodelling, notably in wound repair. The aim of this study was to measure the effects of controlled loading on the release of extracellular matrix protease activities by fibroblasts. Fibroblast populated collagen lattices were subjected to external cyclical loads through a computer controlled unit incorporated into a culture system, a tensioning-Culture Force Monitor. Cyclical loading was compared to untensioned and statically loaded gels (tethered endogenous contraction). Overall changes in a range of protease activities were monitored (chiefly by zymography) as measures of the cyto-mechanical response to these loads. Under static load, 2.5- and 13-fold more matrix metalloproteinase-2 was produced than matrix metalloproteinase-9, at 24 and 48 hours. Total matrix metalloproteinase-9 increased 37 fold on cyclical loading. Total matrix metalloproteinase-3 and urokinase plasminogen activator activities were dramatically reduced on cyclical loading while tissue type plasminogen activator activity was increased. Comparison with cell responses on stiffer substrates (collagen sponges) identified similar matrix metalloproteinase responses to load, but at much reduced levels (4-6 fold matrix metalloproteinase-9 stimulation on loading), showing the importance of matrix compliance to this mechano-response. In conclusion, physiological mechanical loading of fibroblasts in three dimensional collagen lattices elicited complex and substantial changes in matrix modifying proteases. These changes suggest that cells switch between expression of comparable protease activities mainly influencing cell-matrix interactions associated with migration or more generalized extracellular matrix remodelling.

With the rapid development of tissue engineering and gene therapy, collagen-based biomaterials frequently are used as cell transplant devices. In this study we determined the behavior of mouse fibroblasts cultured for up to 6 weeks in control sponges treated by severe dehydration and used commercially as hemostatic agents and in two sponges (DPPA 2 and 3) crosslinked by diphenylphosphorylazide, a method developed in our laboratory. Growth capacity, biosynthetic and proteolytic activities, and matrix reorganization were followed over time in cultures and compared with similar data for fibroblasts in monolayer culture on plastic and in floating or attached collagen gels. Control sponges with and without seeded mouse fibroblasts showed rapid partial denaturation or contraction, weight loss, and severe calcification (13-18% Ca) after 6 weeks. In contrast, the crosslinked sponges showed only slightly decreased size and weight, and the calcification was inhibited (0.2% Ca) in the presence of cells. Mouse fibroblasts seeded on the crosslinked sponge surface at 50,000-200,000 cells/cm(2) progressively penetrated the matrix and proliferated to give the same constant cell density after 3 weeks (around 600,000 cells/sponge). A specific, two- to threefold decrease in collagen synthesis was observed between 1 and 3 or 6 weeks, due mainly to a decrease in the fraction secreted into the medium (25-30% instead of 45-50%). No collagenase 3 activity was detected in the culture medium under any condition or time whereas 25% gelatinase A was found by gelatin zymography to be in an active form in cultures within sponges as compared with less than 10% in monolayers and more than 50% in floating collagen gel. A small amount of gelatinase B was observed after 1 week in sponge cultures and was completely absent thereafter. These results show that the biosynthetic and proteolytic behavior of mouse fibroblasts cultured in crosslinked collagen scaffolds is different from that in monolayers or in floating collagen gels and more similar to that previously described in attached collagen gels.

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