Biological characterization of EDC-crosslinked collagen-hyaluronic acid matrix in dermal tissue restoration

Porous collagen matrices crosslinked with various amounts of hyaluronic acid (HA) by 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) were developed as scaffolds for dermal tissue regeneration. The effect of HA on cells in accordance with HA concentrations in the collagenous matrices was investigated using cultures of fetal human dermal fibroblasts, and the effect of EDC-crosslinked collagen-HA matrix on wound size reduction was also evaluated in vivo. Scanning electron microscopic views of the matrices demonstrated that all of the collagen-HA matrices had interconnected pores with mean diameters of 150-250 microm. An HA matrix retention test showed that the concentration of HA decreased slowly after an initial rapid decrease over 24h. Fetal human dermal fibroblasts adhered well to all of the collagen-based matrices as compared with the Porous polyurethane matrix used as a control. An 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide based proliferation test and the hematoxylin and eosin staining of a 2 week cultured matrix showed that the proliferation of fibroblasts was enhanced on a 9.6% HA contained collagen matrix. No significant difference was in terms of fibroblast migration into the various types of scaffolds as HA content was increased. In vivo testing showed that dermis treated with collagen or collagen-HA matrix was thicker than the control, and epithelial regeneration was accelerated, and collagen synthesis increased. However, no significant effect of HA on wound size reduction was found.     

Modified Dendrimer Cross-Linked Collagen-Based Matrices

Abstract

Dendrimer cross-linking has been achieved with pepsin digested over 80% type-I bovine collagen to create strong hydrogels with good cell compatibility. Herein we investigate the use of commercially available collagen-based products with the dendrimer cross-linking technology. Specifically PureCol^® (PC), a 97% bovine type-I collagen, human collagen (HC) and human extracellular matrix (hECM) were concentrated, and then cross-linked with polypropyleneimine octaamine generation two dendrimers using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry. PC gels with 30 and 20 mg/ml bovine collagen were fabricated, and despite similar concentrations to >80% type-I bovine collagen dendrimer cross-linked gels (CG), PC gels demonstrated increased swelling and decreased stability, as determined with collagenase digestion. The highly purified bovine (PC) and human sourced-collagen (HC) gels were similar in performance, but not as stable as the CG gels, which may correlate to the manufacturer’s collagen purification and storage. Finally, the addition of hECM components to PC to create PC-hECM gels, resulted in a looser gel network, compared to heparinized dendrimer cross- linked bovine >80% type-I collagen gels (CHG). However, all collagen-based gels supported 3T3 fibroblast cell growth over 4 days, indicating these gels may be suitable for tissue-engineering applications.     

Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate

Porous collagen matrices with defined physical, chemical and biological characteristics are interesting materials for tissue engineering. Attachment of glycosaminoglycans (GAGs) may add to these characteristics and valorize collagen. In this study, porous type I collagen matrices were crosslinked using dehydrothermal (DHT) treatment and/or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), in the presence and absence of chondroitin sulphate (CS). EDC covalently attaches CS to collagen. DHT crosslinking preserved a porous matrix structure. However, attachment of CS to DHT-treated matrices using EDC, resulted in collapsed surfaces, CS located only at the matrix exterior. EDC crosslinking resulted in a partial matrix collapse. This could be prevented if crosslinking was carried out in the presence of ethanol. Matrix porosity was then preserved. The presence of CS during EDC crosslinking resulted in covalent immobilization of CS throughout the matrix. The amount of CS incorporated was increased if crosslinking was performed in the presence of ethanol. EDC-crosslinked matrices, with and without CS, had increased denaturation temperatures and decreased free amine group contents. The susceptibility of these matrices towards degradation by proteolytic enzymes was diminished. Immobilized CS increased the water-binding capacity and decreased the denaturation temperature and tensile strength. Immobilized CS bound anti-CS antibodies and was susceptible to chondroitinase ABC digestion, demonstrating its bioavailability. The modified matrices were not cytotoxic as was established using human myoblast and fibroblast culture systems. It is concluded that the use of ethanol during EDC crosslinking, offers an elegant means for the preparation of defined porous collagenous matrices containing bioavailable, covalently attached CS.     

GLIOMA INVASION IN VITRO IS MEDIATED BY CD44–HYALURONAN INTERACTIONS

Invasion is a clinically important problem contributing to mortality and morbidity in patients with gliomas, but the mechanism(s) by which glioma cells invade surrounding brain structures is poorly understood. Various experimental models have been used in attempts to elucidate the process of glioma invasion. An in vitro model which is increasingly being employed involves measurement of the rate of invasion of tumour cells through Matrigel^®—a complex mixture of extracellular matrix components derived from the Engelbroth–Holm–Swarm (EHS) sarcoma. This model has been used to examine the possibility that extracellular hyaluronan (HA) might facilitate the invasive behaviour of human glioma cells. The major component of Matrigel^® is laminin, with smaller amounts of collagen IV, heparan sulphate proteoglycans, entactin, and nidogen, but it lacks HA. In our experiments, we have incorporated HA into Matrigel^® and have measured its effect on the rate of invasion of human glioma cells in a modified Boyden chamber assay system. The incorporation of HA (50–800 mg/cm²) resulted in a dose-dependent increase in invasion. Invasion was enhanced by up to 70 per cent in comparison with HA-free Matrigel^®. Since CD44 is a major HA receptor expressed on gliomas, it might have a role in the HA-mediated facilitation of invasion. This was tested by blocking CD44 with specific antibody, which resulted in a 43 per cent reduction in invasion rate. We conclude that in an in vitro model system, HA enhances invasion of glioma cells and that the mechanism involves a CD44–HA interaction. © 1997 by John Wiley & Sons, Ltd.     

Mesenchymal progenitor cells communicate via alpha and beta integrins with a three-dimensional collagen type I matrix

BACKGROUND/AIMS: The aim of our study was to investigate interactions of mesenchymal progenitor cells (MPCs) with collagen matrices. METHODS: Human bone-marrow-derived MPCs were cultivated in collagen type I gels with and without inhibition of beta(1)-integrin by a specific antibody. Collagen gel contraction, cell morphology, expression of integrin subunits and several genes related to matrix synthesis and turnover as well as MPC differentiation were analyzed over 14 days. RESULTS: Human MPCs markedly contracted free-floating collagen gels. Contraction was nearly completely inhibited by blocking beta(1)-integrin. Cellular morphology was elongated in the absence and mostly round in the presence of the antibody. Expression of integrin alpha(1), alpha(2) and beta(1) subunits showed several changes partly dependent on beta(1)-integrin blocking. Expression of matrix metalloproteinase-1 was elevated irrespective of beta(1)-integrin blocking and tenascin-C was subsequently induced during gel contraction. Spontaneous induction of chondrogenic, osteogenic or adipogenic differentiation was observed neither in the presence nor in the absence of the beta(1)-integrin antibody. CONCLUSION: Our results indicate that the interaction of human MPCs with fibrillar collagen type I involves beta(1)- and alpha-integrin subunits and induces changes in gene expression related to extracellular matrix synthesis and turnover but not differentiation to the chondrogenic, osteogenic or adipogenic phenotype.     

透明质酸对大鼠肺泡巨噬细胞和肺成纤维细胞分泌蛋白酶的影响

目的:研究不同大小透明质酸对大鼠肺泡巨噬细胞和肺成纤维细胞分泌基质金属蛋白酶（MMPs）的诱导作用及对细胞周围胶原收缩和降解的影响。方法:Wistar 大鼠肺泡灌洗获取肺泡巨噬细胞,Wistar 乳鼠肺组织块培养获取肺成纤维细胞;透明质酸（简称HA）分为4个分子量：100 kD、200 kD、500 kD、1 000 kD;将两种细胞混悬液分别与胶原溶液、不同分子量的透明质酸溶液混匀形成胶原凝胶后将凝胶漂浮于培养液中连续培养72 h;每24 h 记录凝胶面积;留取第1个24 h的培养上清液应用酶联免疫吸附法检测MMPs;72 h后收集凝胶,应用分光光度测定法进行羟脯氨酸检测。结果:（1）不同分子量HA都可刺激肺泡巨噬细胞分泌MMP-9、MMP-12和MMP-13,与对照组比差异显著(P<0.05);其中以200 kD HA作用最强。（2）不同分子量HA都可刺激肺成纤维细胞分泌MMP-1、MMP-9和MMP-13,与对照组比较,P<0.05;其中以100、200 kD HA作用最为显著。（3）凝胶形成后24 h,100、200、500 kD HA使肺泡巨噬细胞凝胶面积明显缩小,与对照组比较有显著差异（P<0.05),并且这种作用一直持续到72 h。（4）凝胶形成后24 h, 100、200、500 kD的HA使肺成纤维细胞凝胶面积明显缩小,与1 000 kD HA组及对照组比较有显著差异(P<0.05));72 h后,100、200 kD HA使凝胶面积进一步缩小,与500、1 000 kD HA组及对照组比较,P<0.05。（5）4种分子量的HA与肺泡巨噬细胞共同孵育72h后,凝胶中羟脯氨酸含量与对照组比较均有显著减少,其中100、200 kD HA组羟脯氨酸含量降低最显著(P<0.05)。（6）4种分子量的HA与成纤维细胞共同孵育72h后,凝胶中羟脯氨酸含量与对照组比较均显著减少,且不同分子量之间差异显著(P<0.05)。结论：（1）HA可以刺激大鼠肺泡巨噬细胞和肺成纤维细胞分泌多种与胶原降解相关的MMPs;其中200 kD HA作用最强。（2）HA可以促进两种细胞凝胶收缩,以100、200、500 kD的HA作用最显著。（3）HA可以降解细胞凝胶中的胶原,使其羟脯氨酸含量减低,以100、200 kD HA作用最强。     

Effects of hydroxyapatite on endothelial network formation in collagen/fibrin composite hydrogels in vitro and in vivo

Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3-D collagen/fibrin (COL/FIB) composite hydrogels. However, the ability to form endothelial networks is lost when such gels are allowed to compact via cell-mediated remodeling. In this study, we created co-cultures of human EC and human MSC in both constrained and unconstrained COL/FIB matrices and systematically added nanoparticulate hydroxyapatite (HA, 0–20 mg ml⁻¹), a bone-like mineral that has been shown to have pro-vasculogenic effects. Constructs cultured for 7 days were assayed for gel compaction, vascular network formation, and mechanical properties. In vitro, robust endothelial network formation was observed in constrained COL/FIB constructs without HA, but this response was significantly inhibited by addition of 5, 10, or 20 mg ml⁻¹ HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs but was rescued by 1.25 or 2.5 mg ml⁻¹ HA, while higher levels again inhibited vasculogenesis. HA inhibited gel compaction in a dose-dependent manner, which was not correlated to endothelial network formation. HA affected initial stiffness of the gels, but gel remodeling abrogated this effect. Subcutaneous implantation of COL/FIB with 0, 2.5 or 20 mg ml⁻¹ HA in the mouse resulted in increased perfusion at the implant site, with no significant differences between materials. Histology at day 7 showed both host and human CD31-stained vasculature infiltrating the implants. These findings are relevant to the design of materials and scaffolds for orthopedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.     

A collagen-based scaffold for a tissue engineered human cornea: physical and physiological properties

Stabilized collagen-glycosaminoglycan scaffolds for tissue engineered human corneas were characterized. Hydrated matrices were constructed by blending type I collagen with chondroitin sulphates (CS), with glutaraldehyde crosslinking. A corneal keratocyte cell line was added to the scaffolds with or without corneal epithelial and endothelial cells. Constructs were grown with or without ascorbic acid. Wound-healing was evaluated in chemical-treated constructs. Native, noncrosslinked gels were soft with limited longevity. Crosslinking strengthened the matrix yet permitted cell growth. CS addition increased transparency. Keratocytes grown within the matrix had higher frequencies of K+ channel expression than keratocytes grown on plastic. Ascorbic acid increased uncrosslinked matrix degradation in the presence of keratocytes, while it enhanced keratocyte growth and endogenous collagen synthesis in crosslinked matrices. Wounded constructs showed recovery from exposure to chemical irritants. In conclusion, this study demonstrates that our engineered, stabilized matrix is well-suited to function as an in vitro corneal stroma.     

The effect of bioactive hydrogels on the secretion of extracellular matrix molecules by valvular interstitial cells

Valvular interstitial cells (VICs) were encapsulated in enzymatically degradable, crosslinked hydrogels formed from hyaluronic acid (HA) and poly(ethylene glycol) (PEG) macromolecular monomers. Titration of PEG with HA allowed for the synthesis of gels with a broad compositional spectrum, leading to a range of degradation behavior upon exposure to bovine testes hyaluronidase. The rate of mass loss and release of HA fragments from the copolymer gels depended on the PEG content of the network. These hydrogels were shown to have the dual function of permitting the diffusion of ECM elaborated by 3D cultured VICs and promoting the development of a specific matrix composition. Initial cleavage of hydrogel crosslinks, prior to network mass loss, permit the diffusion of collagen, while later stages of degradation promote elastin elaboration and suppress collagen production due to HA fragment release. Exogenous HA delivery through the cell culture media further demonstrated the utility of delivered HA on manipulating the secretory properties of encapsulated VICs.     

In vitro reconstruction of human dermal equivalent enriched with endothelial cells

Experiences coming from many cell-culture studies has brought about the concept that tissue and organ reconstruction should be performed in a three-dimensional environment as it normally occurs in vivo. As far as endothelial cell culture is concerned, it has been shown that angiogenesis can be successfully achieved only when cells are cultured in the presence of collagen-based matrices or basal membrane substrates. The aim of the present investigation is to demonstrate that human umbilical vein endothelial cells (HUVEC) can be grown and differentiated on an artificial dermis obtained by fibroblasts cultured on hyaluronic acid-based scaffolds. For this purpose, we have cultured HUVEC, retrieved by collagenase digestion of perfused human umbilical vein either alone and with fibroblast at 1/1 ratio into HYAFF-11 non-woven mesh. Cultures were maintained for up to 3 weeks. Samples were taken at different time points within this period for the MTT proliferation test and for immunohistochemical analysis. Our results demonstrate that hyaluronan-based biomaterials (HYAFF-11 NW mesh) represent a suitable substrate for HUVEC adhesion, proliferation and reorganization in microcapillary network.     

Characterization of collagen matrices crosslinked using microbial transglutaminase

In search of a new approach for crosslinking collagen-based biomaterials, we examined the effect of microbial transglutaminase (MTGases) as a crosslinking reagent on collagenous matrices made from porcine type I collagen. As the results revealed, MTGase exhibited a crosslinking action that raised the viscosity of the collagen solution. Matrices crosslinked with MTGase at the low pH values of pH 3 and 4 exhibited higher tensile strengths than those at high pH values. In comparison with untreated matrices, the denaturation temperatures of the corresponding matrices shifted toward higher temperatures. These enzyme-catalyzed crosslinked matrices were proven by MTT assay to be non-cytotoxic. In conclusion, this enzymatic method of using MTGase provides an alternative potential way for crosslinking collagen-based matrices.     

Growth promoting substrates for human dermal fibroblasts provided by artificial extracellular matrices composed of collagen I and sulfated glycosaminoglycans

The application of native extracellular matrix (ECM) components is a promising approach for biomaterial design. Here, we investigated artificial ECM (aECM) consisting of collagen I (coll) and the glycosaminoglycans (GAGs) hyaluronan (HA) or chondroitin sulfate (CS). Additionally, GAGs were chemically modified by the introduction of sulfate groups to obtain low-sulfated and high-sulfated GAG derivatives. Sulfate groups are expected to bind and concentrate growth factors and improve their bioactivity. In this study we analyzed the effect of aECM on initial adhesion, proliferation, ECM synthesis and differentiation of human dermal fibroblasts (dFb) within 8-48 h. We show that initial adhesion and cell proliferation of dFb progressively increased in a sulfate dependent manner. In contrast, synthesis of ECM components coll and HA was decreased on high-sulfated aECM coll/HA3.0 and coll/CS3.1. Furthermore, the matrix metallo-proteinase-1 (MMP-1) was down-regulated on coll/HA3.0 and coll/CS3.1 on mRNA and protein level. The fibroblast differentiation marker α-smooth muscle actin (αSMA) is not affected by aECM on mRNA level. Artificial ECM consisting of coll and high-sulfated GAGs proves to be a suitable biomaterial for dFb adhesion and proliferation that induces a "proliferative phenotype" of dFb found in the early stages of cutaneous wound healing.     

Mouse fibroblasts in long-term culture within collagen three-dimensional scaffolds: influence of crosslinking with diphenylphosphorylazide on matrix reorganization, growth, and biosynthetic and proteolytic activities

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.     

Collagen for bone tissue regeneration

In the last decades, increased knowledge about the organization, structure and properties of collagen (particularly concerning interactions between cells and collagen-based materials) has inspired scientists and engineers to design innovative collagen-based biomaterials and to develop novel tissue-engineering products. The design of resorbable collagen-based medical implants requires understanding the tissue/organ anatomy and biological function as well as the role of collagen's physicochemical properties and structure in tissue/organ regeneration. Bone is a complex tissue that plays a critical role in diverse metabolic processes mediated by calcium delivery as well as in hematopoiesis whilst maintaining skeleton strength. A wide variety of collagen-based scaffolds have been proposed for different tissue engineering applications. These scaffolds are designed to promote a biological response, such as cell interaction, and to work as artificial biomimetic extracellular matrices that guide tissue regeneration. This paper critically reviews the current understanding of the complex hierarchical structure and properties of native collagen molecules, and describes the scientific challenge of manufacturing collagen-based materials with suitable properties and shapes for specific biomedical applications, with special emphasis on bone tissue engineering. The analysis of the state of the art in the field reveals the presence of innovative techniques for scaffold and material manufacturing that are currently opening the way to the preparation of biomimetic substrates that modulate cell interaction for improved substitution, restoration, retention or enhancement of bone tissue function.     

Functional biopolymer-based matrices for modulation of chronic wound enzyme activities

Collagen, collagen/hyaluronic acid (HA) and collagen/HA/chitosan (CS) sponges loaded with epigallocatechin gallate (EGCG), catechin (CAT) and gallic acid (GA) were developed and evaluated as active chronic wound dressings. Their physico-mechanical properties, biostability, biocompatibility and ability to inhibit in vitro myeloperoxidase (MPO) and collagenase—major enzymes related with the persistent inflammation in chronic wounds—were investigated as a function of the biopolymer composition and the polyphenolic compound used. The results demonstrated that the molecular weight of HA influences significantly the bulk properties of the obtained materials: higher elastic modulus, swelling ability and biostability against collagenase were measured when HA with higher molecular weights (830 and 2000 kDa) were added to the collagen matrices. The addition of CS and the polyphenols increased further the biostability of the sponges. Preliminary in vitro tests with fibroblasts revealed that the cells were able to adhere to all sponges. Cell viability was not affected significantly by the addition of the polyphenols; however, the presence of CS or high molecular weight HA in the sponge composition was associated with lower cellular viability. Finally, all specimens containing polyphenols efficiently inhibited the MPO activity. The highest inhibition capacity was observed for EGCG (IC₅₀ = 15 ± 1 μM) and it was coupled to the highest extent of binding to the biopolymers (>80%) and optimal release profile from the sponges that allowed for prolonged (up to 3–5 days) effects.     

Development of tailor-made collagen-glycosaminoglycan matrices: EDC/NHS crosslinking, and ultrastructural aspects

The many biocharacteristics of glycosaminoglycans (GAGs) make them valuable molecules to be incorporated in collagenous biomaterials. To prepare tailor-made collagen-GAG matrices with a well-defined biodegradability and (bioavailable) GAG content, the crosslinking conditions have to be controlled. Additionally, the ultrastructural location of GAGs in engineered substrates should resemble that of the application site. Using chondroitin sulfate (CS) as a model GAG, these aspects were evaluated. The methodology was then applied for other GAGs. CS was covalently attached to collagen using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). A maximum of about 155 mg CS/g matrix could be immobilized. CS incorporation and bioavailability, as evaluated by interaction with specific antibodies and glycosidases, was dependent on the molar ratio EDC:carboxylic groups of CS. The denaturation temperature could be modulated from 61 to 85 degrees C. The general applicability of EDC/NHS for immobilizing GAGs was demonstrated with dermatan sulfate, heparin, and heparan sulfate. These matrices revealed comparable physico-chemical characteristics, biodegradabilities, and preserved bioavailable GAG moieties. At the ultrastructural level, GAGs appeared as discrete, electron-dense filaments, each filament representing a single GAG molecule. Distribution was independent of GAG type. They were observed throughout the matrix fibers and at the outer sites, and located, either parallel or orthogonally, at the periphery of individual collagen fibrils. Compositional and ultrastructural similarity between matrices and tissue structures like cartilage and basement membranes can be realized after attachment of specific GAG types. It is concluded that EDC/NHS is generally applicable for attachment of GAGs to collagen. Modulation of crosslinking conditions provides matrices with well-defined GAG contents, and biodegradabilities. Ultrastructural similarities between artificially engineered scaffolds and their possible application site may favor the use of specific collagen-GAG matrices in tissue engineering.     

Network connectivity, mechanical properties and cell adhesion for hyaluronic acid/PEG hydrogels

The study aimed to explore the influence of the network architecture on the mechanical properties and degradability of HA/PEG gels, and to highlight the relationship between Young's modulus and cell colonization with a selected architecture. Three different families of hyaluronic acid (HA)-based photopolymerized PEG diacrylate (PEGDA) hydrogels were compared, using different concentrations and molecular weights (64 and 234?kDa) of HA: semi-IPNs containing native HA in a PEG network (type I gels); co-networks obtained using thiolated HA as chain transfer agent during PEGDA polymerization (type II gels); co-networks obtained from the in situ preparation of a macromonomer derived from the Michael-type addition of thiolated HA on PEGDA (type III gels). From a comparative study of rheological properties and enzymatic degradability, type II gels were selected for a further study aiming to link their mechanical properties to cell spreading. Employing RGD-functionalized materials, Young's moduli were measured via AFM nanoindentation while the cell spreading behavior was quantitatively evaluated by monitoring morphology and metabolic activity (MTS assay) of L929 fibroblasts. By revealing a clear relation between increasing modulus and increasing cell spreading/proliferation, the study showed the possibility to fine tune the cell/material interactions with appropriate reactive processing techniques.     

Cross-linked type I and type II collagenous matrices for the repair of full-thickness articular cartilage defects--a study in rabbits

The physico-chemical properties of collagenous matrices may determine the tissue response after insertion into full-thickness articular cartilage defects. In this study, cross-linked type I and type II collagen matrices, with and without attached chondroitin sulfate, were implanted into full-thickness defects in the femoral trochlea of adolescent rabbits. The tissue response was evaluated 4 and 12 weeks after implantation by general histology and two semi-quantitative histological grading systems. Four weeks after implantation, type I collagenous matrices were completely filled with cartilage-like tissue. By contrast, type II collagenous matrices revealed predominantly cartilaginous tissue only at the superficial zone and at the interface of the matrix with the subchondral bone, leaving large areas of the matrix devoid of tissue. Attachment of chondroitin sulfate appeared to promote cellular ingrowth and cartilaginous tissue formation in both types of collagen matrices. Twelve weeks after implantation, the differences between the matrices were less pronounced. The deep parts of the subchondral defects were largely replaced by new bone with a concomitant degradation of the matrices. The original cartilage contours in defects with type I collagen-based matrices were repaired with fibro-cartilaginous tissue. Defects containing type II matrices showed an increase in the amount of superficial cartilage-like tissue. The original contour, however, was not completely restored in all animals, occasionally leaving a central depression or fissure. It is concluded that different types of collagen matrices induce different tissue responses in full-thickness articular cartilage defects. Type I collagen-based matrices are superior to guide progenitor cells from a subchondral origin into the defect. In type II collagen-based matrices cell migration is less, but invading cells are directed into a chondrocyte phenotype. Based on these observations it is suggested that a composite matrix consisting of a deep layer of type I collagen and a more superficial layer of type II collagen may be the matrix of choice for cartilage regeneration.     

Collagen Gel Anisotropy Measured by 2-D Laser Trap Microrheometry

Collagen gels can serve as biomaterials ideal for tissue equivalents, especially if they are remodeled to have fibril anisotropy mimicking native tissue. Type I collagen gel remodeling was studied microscopically to investigate the changes caused by fibroblasts in collagen gel structures, with and without the growth factors PDGF-BB and TGF-β1. A bidirectional laser trap microrheometry technique was developed that revealed a high degree of local heterogeneity and anisotropy in the structure of the collagen gels during active fibroblast contraction. The use of the growth factors increased not only the gel anisotropy, but the heterogeneity as well, indicating further changes in the collagen fibril orientations. This work shows the ability to influence the remodeling capabilities of fibroblasts by using growth factors in order to begin to elucidate the changes in the local mechanical environment of contracting collagen gels. We present this experimental technique as a method for probing changes in the fibroblast-driven anisotropy of collagen gels as a basis for understanding microstructural tissue organization important in the development of collagen-based tissue equivalents.