collagen-GAG Matrix Research Articles

The Use of Collagen-Glycosaminoglycan Biodegradable Matrix (Integra®) in the Management of Neck Postburn Hypertrophic Scars and Contractures

Glycosaminoglycan (GAG) is a chain-like disaccharide that is linked to a polypeptide core to connect two collagen fibrils/fibers and provide the intermolecular force in a Collagen-GAG matrix which can be a valuable treatment of post-burn contractures and hypertrophic scars, which remain a challenge to reconstructive surgery. The face and neck contractures are the most difficult sites to treat. This article is meant to discuss our clinical experience in using collagen-glycosaminoglycan biodegradable matrix (Integra® Integra Lifesciences Corporation, Plainsboro, NJ, USA) to reconstruct defects created by excision of contracted areas from the neck and lower face areas. Between 2009 and 2011, we had 11 patients that underwent Integra reconstructive procedures. The mean follow-up period was 18 months. For all the patients, the intake of the Integra dermal regeneration template was 100%, even if one patient developed a minor infection treated with appropriate antibiotics. The patients are very satisfied with the result. A minor problem was a small difference in skin color, but this inconvenience was compensated by good skin elasticity.

Structure of collagen-glycosaminoglycan matrix and the influence to its integrity and stability.

Glycosaminoglycan (GAG) is a chain-like disaccharide that is linked to polypeptide core to connect two collagen fibrils/fibers and provide the intermolecular force in Collagen-GAG matrix (C-G matrix). Thus, the distribution of GAG in C-G matrix contributes to the integrity and mechanical properties of the matrix and related tissue. This paper analyzes the transverse isotropic distribution of GAG in C-G matrix. The angle of GAGs related to collagen fibrils is used as parameters to qualify the GAGs isotropic characteristic in both 3D and 2D rendering. Statistical results included that over one third of GAGs were perpendicular directed to collagen fibril with symmetrical distribution for both 3D matrix and 2D plane cross through collagen fibrils. The three factors tested in this paper: collagen radius, collagen distribution, and GAGs density, were not statistically significant for the strength of Collagen-GAG matrix in 3D rendering. However in 2D rendering, a significant factor found was the radius of collagen in matrix for the GAGs directed to orthogonal plane of Collagen-GAG matrix. Between two cross-section selected from Collagen-GAG matrix model, the plane cross through collagen fibrils was symmetrically distributed but the total percentage of perpendicular directed GAG was deducted by decreasing collagen radius. There were some symmetry features of GAGs angle distribution in selected 2D plane that passed through space between collagen fibrils, but most models showed multiple peaks in GAGs angle distribution. With less GAGs directed to perpendicular of collagen fibril, strength in collagen cross-section weakened. Collagen distribution was also a factor that influences GAGs angle distribution in 2D rendering. True hexagonal collagen packaging is reported in this paper to have less strength at collagen cross-section compared to quasi-hexagonal collagen arrangement. In this work focus is on GAGs matrix within the collagen and its relevance to anisotropy.

Aligned collagen-GAG matrix as a 3D substrate for Schwann cell migration and dendrimer-based gene delivery.

The development of artificial off-the-shelf conduits that facilitate effective nerve regeneration and recovery after repair of traumatic nerve injury gaps is of fundamental importance. Collagen-glycosaminoglycan (GAG) matrix mimicking Schwann cell (SC) basal lamina has been proposed as a suitable and biologically rational substrate for nerve regeneration. In the present study, we have focused on the permissiveness of this matrix type for SC migration and repopulation, as these events play an essential role in nerve remodeling. We have also demonstrated that SCs cultured within collagen-GAG matrix are compatible with non-viral dendrimer-based gene delivery, that may allow conditioning of matrix-embedded cells for future gene therapy applications.

Return of Motor Function After Repair of a 3-cm Gap in a Rabbit Peroneal Nerve

The purpose of this study was to evaluate the motor nerve recovery in a rabbit model after repair of a 3-cm gap in the peroneal nerve with a conduit filled with a collagen-GAG (glycosaminoglycan) matrix and compare the results with those after reconstruction with an autograft or an empty collagen conduit. Forty-two male New Zealand rabbits were divided into three experimental groups. In each group, a unilateral 3-cm peroneal nerve defect was repaired with a nerve autograft, an empty collagen conduit, or a conduit filled with a collagen-GAG matrix. At six months, nerve regeneration was evaluated on the basis of the compound muscle action potentials, maximum isometric tetanic force, and wet muscle weight of the tibialis anterior muscle as well as nerve histomorphometry. The autograft group had significantly better motor recovery than the conduit groups. The empty collagen conduits and conduits filled with the collagen-GAG matrix led to results that were similar to each other. On the basis of this rabbit model, autologous nerve grafting remains the gold standard in the reconstruction of 3-cm segmental motor nerve defects. Segmental motor nerve defects should be reconstructed with autograft nerves. The use of a collagen conduit filled with a collagen-GAG matrix for motor nerve reconstruction should be limited until additional animal studies are performed.

Release of growth factors from a reinforced collagen GAG matrix supplemented with platelet rich plasma: Influence on cultured human meniscal cells

Damage to meniscal cartilage has been strongly linked to accelerated articular wear and consequently to osteoarthritis. Damage might be ameliorated by delivery of growth factors from platelet rich plasma (PRP) via a fiber reinforced collagen matrix designed for meniscal repair. PRP composition, release of growth factors, and influence on meniscal cell growth and gene expression were investigated. PRP was prepared using Harvest Smartprep (HS-PRP), Cascade Fibrinet (CF-PRP), and a simple centrifuge protocol (DC-PRP) from four donors each. CF-PRP had the highest ratio of platelets, with very few other blood cell types. HS-PRP had the highest total number of platelets but also contained high levels of red and white blood cells. Absorbed to collagen matrices HS-PRP released the highest levels of TGF-β1 and PDGF-AB with DC-PRP the most IGF-1. Cumulative release from collagen matrix was 48 ng/cm(3) IGF-1, 96 ng/cm(3) TGF-β1, and 9.6 ng/cm(3) PDGF-AB. Collagen matrix with PRP was able to increase meniscal cell number above peripheral whole blood and up-regulated gene expression of Aggrecan, Collagen type I (α1), and Elastin (3.3 ± 0.8-fold, 2.9 ± 0.6-fold, 4.0 ± 1.4-fold, respectively). Demonstrating that PRP combined with fiber reinforced collagen matrix could influence meniscal cells and might be of use for treating meniscal defects.

Improved Healing of Large Segmental Defects in the Rat Femur by Reverse Dynamization in the Presence of Bone Morphogenetic Protein-2

Large segmental defects in bone do not heal well and present clinical challenges. This study investigated modulation of the mechanical environment as a means of improving bone healing in the presence of bone morphogenetic protein (BMP)-2. Although the influence of mechanical forces on the healing of fractures is well established, no previous studies, to our knowledge, have described their influence on the healing of large segmental defects. We hypothesized that bone-healing would be improved by initial, low-stiffness fixation of the defect, followed by high-stiffness fixation during the healing process. We call this reverse dynamization. A rat model of a critical-sized femoral defect was used. External fixators were constructed to provide different degrees of stiffness and, importantly, the ability to change stiffness during the healing process in vivo. Healing of the critical-sized defects was initiated by the implantation of 11 μg of recombinant human BMP (rhBMP)-2 on a collagen sponge. Groups of rats receiving BMP-2 were allowed to heal with low, medium, and high-stiffness fixators, as well as under conditions of reverse dynamization, in which the stiffness was changed from low to high at two weeks. Healing was assessed at eight weeks with use of radiographs, histological analysis, microcomputed tomography, dual x-ray absorptiometry, and mechanical testing. Under constant stiffness, the low-stiffness fixator produced the best healing after eight weeks. However, reverse dynamization provided considerable improvement, resulting in a marked acceleration of the healing process by all of the criteria of this study. The histological data suggest that this was the result of intramembranous, rather than endochondral, ossification. Reverse dynamization accelerated healing in the presence of BMP-2 in the rat femur and is worthy of further investigation as a means of improving the healing of large segmental bone defects. These data provide the basis of a novel, simple, and inexpensive way to improve the healing of critical-sized defects in long bones. Reverse dynamization may also be applicable to other circumstances in which bone-healing is problematic.

The Effect of Collagen Nerve Conduits Filled with Collagen-Glycosaminoglycan Matrix on Peripheral Motor Nerve Regeneration in a Rat Model

Bioabsorbable unfilled synthetic nerve conduits have been used in the reconstruction of small segmental nerve defects with variable results, especially in motor nerves. We hypothesized that providing a synthetic mimic of the Schwann cell basal lamina in the form of a collagen-glycosaminoglycan (GAG) matrix would improve the bridging of the nerve gap and functional motor recovery. A unilateral 10-mm sciatic nerve defect was created in eighty-eight male Lewis rats. Animals were randomly divided into four experimental groups: repair with reversed autograft, reconstruction with collagen nerve conduit (1.5-mm NeuraGen, Integra LifeSciences), reconstruction with collagen nerve conduit filled with collagen matrix, and reconstruction with collagen nerve conduit filled with collagen-GAG (chondroitin-6-sulfate) matrix. Nerve regeneration was evaluated at twelve weeks on the basis of the compound muscle action potential, maximum isometric tetanic force, and wet muscle weight of the tibialis anterior muscle, the ankle contracture angle, and nerve histomorphometry. The use of autograft resulted in significantly better motor recovery compared with the other experimental methods. Conduit filled with collagen-GAG matrix demonstrated superior results compared with empty conduit or conduit filled with collagen matrix with respect to all experimental parameters. Axon counts in the conduit filled with collagen-GAG matrix were not significantly different from those in the reversed autograft at twelve weeks after repair. The addition of the synthetic collagen basal-lamina matrix with chondroitin-6-sulfate into the lumen of an entubulation repair significantly improved bridging of the nerve gap and functional motor recovery in a rat model. Use of a nerve conduit filled with collagen-GAG matrix to bridge a motor or mixed nerve defect may result in superior functional motor recovery compared with commercially available empty collagen conduit. However, nerve autograft remains the gold standard for reconstruction of a segmental motor nerve defect.

Improving the Pancreatic Islet Graft Structure and Function: Application of a Novel Bioengineered Composite Scaffold

Introduction: Transplantation of the insulin-producing islet cells of the pancreas has been a promising strategy for management of type 1 diabetes. However, shortage of islet donors, and poor survival of the islets after transplantation are some of the main challenges of this procedure. Enzymatic digestion of the pancreas in the process of islet isolation results in the loss of peri-insular extracellular matrix (ECM) and peri-vascular basement membrane of the islets that can significantly disrupt the ECM-mediated interactions and result in apoptosis. Naturally a collagen matrix provides for a viable environment in which both stromal and islet cells can survive. However this matrix is mainly biodegradable and subject to gradual disintegration and contraction after transplantation. We have previously shown that a fibroblast populated collagen matrix (FPCM) significantly improves islet cell viability and glucose responsiveness and reduces the marginal islet mass required for hyperglycemia reversal in diabetic mice. Fibroblasts were incorporated to provide a favorable support for islets by producing various growth and angiogenic factors, while maintaining the integrity of the collagen matrix. Although this composite was able to promote the islet graft survival and function, it was still prone to gradual biodegradation. For this reason, we designed a novel bioengineered crosslinked-interpenetrating network of glycosaminoglycan (GAG) and type I collagen (Collagen-GAG matrix, CGM). When populated with fibroblasts (FP-CGM), it would provide an optimal matrix biomimetic with reduced contraction and enhanced mechanical strength for the transplanted islets. Methods: Pancreatic islets and dermal fibroblasts were obtained from Balb/c mice. Islets (50 islets per well in 48-well plates) were cultured in medium (2D), acellular collagen matrix (CM), FPCM, acellular CGM and FP-CGM composites. In order to prepare the FPCM and FP-CGM composites, 200,000 fibroblasts per 100μl of the gel were used. The viability of the islets within composites was assessed by a Live/Dead assay kit on days 1, 15 and 30 post culture. Islets were retrieved on days 1, 15 and 30 post culture and paraffin-embedded sections were stained for insulin, glucagon, caspase-3 and Ki67 and the β-cell proliferation rate was calculated. Retrieved islets were evaluated for glucose-stimulated insulin secretion. Finally, the expression of the islet key specific genes including insulin, PDX1, GLUT2, Pax4, and Pax6 were examined before and after culture at different conditions using reverse transcriptase-PCR analysis. Results: Our preliminary findings have demonstrated that our matrix is non-toxic to both fibroblasts and islets and able to withstand contraction in vitro: Live and Dead assay showed that islets perfectly survive within the new composite matrix. Histology revealed reductions in graft contraction and fibroblast cellularity, increased insulin and glucagon production, and decreased apoptosis rate compared to naked islets in 2D system. Islet morphology remained intact within the composite FP-CGM. PCR analysis showed high expression of islets key genes when islets are embedded within the proposed matrix. Conclusions: We have identified that the use of a crosslinked collagen-GAG graft has the potential to improve graft survival, improving the post-surgical outcome of the transplanted islets.

Functional improvement and neurogenesis after collagen-GAG matrix implantation into surgical brain trauma

Surgical or traumatic brain injury often leads to loss of cerebral parenchyma but there is as yet no clinically effective strategy for neural regeneration. Collagen glycosaminoglycan (collagen-GAG, CG) scaffolds have previously been used in many tissues in vivo but have never been utilized in the brain. Using an animal model, we investigated the effects of the implantation of CG scaffold matrix following surgical brain trauma. Results indicated that implantation of CG scaffold could significantly promote functional recovery following surgical brain trauma. The CG scaffold was found to facilitate proliferation, differentiation and migration of endogenous neural precursor cells (NPCs) both in the intra-matrix zone (IMZ) and lesion boundary zone (LBZ). The tissue concentration of brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF) in the cortex demonstrated a sustained increase after implantation of CG scaffold following surgical brain trauma. These results suggest that the utilization of CG scaffolds can be considered as a potential clinical strategy for tissue regeneration and functional recovery after brain injury.

Restructuring skin equivalents with human hair follicle orscs and fibroblasts

Background: The bulge region, a portion of the outer root sheath (ORS), contains the hair follicle stem cells, which show multipotency to differentiate into almost all epithelial cell types. To produce an improved quality skin equivalent (SE), we established a rapid, easy and effective three-dimensional SE model on the basis of human dermal fibroblasts, collagen-GAG matrix (DE) and ORS cells (ORSCs).

Lysyl Oxidase-Like and Lysyl Oxidase Are Present in the Dermis and Epidermis of a Skin Equivalent and in Human Skin and Are Associated to Elastic Fibers

Elastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model, keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association with elastin-positive materials within the SE and human skin, though interaction with collagen could not be discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes should play a role in post-translational modification of yet unknown substrates.

Degradation of a collagen–chondroitin-6-sulfate matrix by collagenase and by chondroitinase

Highly porous, type I collagen–chondroitin-6-sulfate (collagen–GAG) scaffolds, produced by freeze-drying techniques, have proven to be of value as implants to facilitate the regeneration of certain tissues. The objective of this project was to evaluate changes in the microstructure and mechanical properties of selected collagen–GAG scaffolds as they degrade in an in vitro model system. Environmental scanning electron microscopy and video imaging demonstrated that collagenase degradation caused strut erosion through the creation of 1–3 μm diameter micropits within a 2-h period, leading to eventual removal of strut material and strut breakage. Loss of microstructural topography may have been due to gelatinization when collagen was cleaved by collagenase. Chondroitinase degradation of GAG resulted in swelling of the struts, causing the pores to become smaller and rounder. The compressive modulus of the collagen–GAG matrix decreased when degraded by collagenase, but remained unchanged when degraded by chondroitinase. Carbodiimide-cross-linked matrices were found to have a higher cross-link density, a higher compressive stiffness and a greater resistance to collagenase and chondroitinase, compared to non-cross-linked controls and matrices that were cross-linked by the dehydrothermal process. This investigation provides information that can be used to design collagen–GAG scaffolds with desired compressive stiffness and degradation rate to collagenase and chondroitinase.

Micromechanics of Fibroblast Contraction of a Collagen–GAG Matrix

The contractile force developed by fibroblasts has been studied by measuring the macroscopic contraction of porous collagen–GAG matrices over time. We have identified the microscopic deformations developed by individual fibroblasts which lead to the observed macroscopic matrix contraction. Observation of live cells attached to the matrix revealed that matrix deformation occurred as a result of cell elongation. The time dependence of the increase in average fibroblast aspect ratio over time corresponded with macroscopic matrix contraction, further linking cell elongation and matrix contraction. The time dependence of average fibroblast aspect ratio and macroscopic matrix contraction was found to be the result of the stochastic nature of cell elongation initiation and of the time required for cells to reach a final morphology (2–4 h). The proposed micromechanics associated with observed buckling or bending of individual struts of the matrix by cells may, in part, explain the observation of a force plateau during macroscopic contraction. These findings indicate that the macroscopic matrix contraction measured immediately following cell attachment is related to the extracellular force necessary to support cell elongation, and that macroscopic time dependence is not directly related to microscopic deformation events.

Fibroblast contraction of a collagen–GAG matrix

Contractile cells, found in wounded or diseased tissues, are associated with the formation of scar tissue. The complexity of contraction in vivo has led to the development of models of contraction by cells in vitro. In this work, a device was developed which quantitatively measured the contractile force developed by fibroblasts seeded into a collagen–glycosaminoglycan porous matrix in vitro. This device differed from most of those previously described in that it directly transferred cellular contractile force to the force transducer (a cantilever beam) and that it used a porous matrix rather than a collagen gel. The data for the increase in contractile force with time were fit to a mathematical equation using two fitting parameters. Data were then compared using the fitting parameters and the cell density. A study of the effect of cell density on the contractile force resulted in a linearly proportional relationship. Subsequent normalization of force by cell density or number resulted in a value of contractile force per cell, 1 nN, that was independent of cell density. The time for the contractile force to develop was also independent of cell density. These results suggest that, in this system, cells develop contractile force individually, irrespective of the force generated by surrounding cells.

Near-terminus axonal structure and function following rat sciatic nerve regeneration through a collagen-GAG matrix in a ten-millimeter gap.

The objectives of this study were to evaluate the regenerated axon structure at near-terminal locations in the peroneal and tibial branches 1 year following implantation of several tubular devices in a 10-mm gap in the adult rat sciatic nerve and to determine the extent of recovery of selected sensory and motor functions. The devices were collagen and silicone tubes implanted alone or filled with a porous collagen-glycosaminoglycan matrix. Intact contralateral nerves and autografts were used as controls. Nerves were retrieved at 30 and 60 weeks postoperatively for histological evaluation of the number and diameter of regenerated axons proximal and distal to the gap and in the tibial and peroneal nerve branches, near the termination point. Several functional evaluation methods were employed: gait analysis, pinch test, muscle circumference, and response to electrical stimulation. A notable finding was that the matrix-filled collagen tube group had a significantly greater number of large-diameter myelinated axons (> or =6 microm in diameter) in the distal nerve branches than any other group, including the autograft group. These results were consistent with previously reported electrophysiological measurements that showed that the action potential amplitude for the A fibers in the matrix-filled collagen tube group was greater than for the autograft control group. Functional testing revealed the existence of both sensory and motor recovery following peripheral nerve regeneration through all devices; however, the tests employed in this study did not show differences among the groups with regeneration. Electrical stimulation in vivo showed that threshold parameters to elicit muscle twitch were the same for reinnervating and control nerves. The investigation is of importance in showing for the first time the superiority of a specific fully resorbable off-the-shelf device over an autograft for bridging gaps in peripheral nerve, with respect to the near-terminus axonal structure.

Connective tissue response to tubular implants for peripheral nerve regeneration: the role of myofibroblasts.

The presence of contractile cells, their organization around regenerating nerve trunks, and the hypothetical effect of these organized structures on the extent of regeneration across a tubulated 10-mm gap in the rat sciatic nerve were investigated. Collagen and silicone tubes were implanted both empty and filled with a collagen-glycosaminoglycan (GAG) matrix. Nerves were retrieved at 6, 30, and 60 weeks postoperatively and time-dependent values of the nerve trunk diameter along the tubulated length were recorded. The presence of myofibroblasts was identified immunohistochemically using a monoclonal antibody to alpha-smooth muscle actin. Myofibroblasts were circumferentially arranged around the perimeter of regenerated nerve trunks, forming a capsule which was about 10 times thicker in silicone tubes than in collagen tubes. The nerve trunk diameter that formed inside collagen tubes was twice as large as that inside silicone tubes. In contrast, the collagen-GAG matrix had a relatively small effect on capsule thickness or diameter of regenerate. It was hypothesized that the frequency of successful bridging by axons depends on the balance between two competitive forces: the axial forces generated by the outgrowth of axons and nonneuronal cells from the proximal stump and the constrictive, circumferential forces imposed by the contractile tissue capsule that promote closure of the wounded stumps and prevent axon elongation. Because the presence of the collagen-GAG matrix has enhanced greatly the recovery of normal function of regenerates in silicone tubes, it was hypothesized that it accelerated axonal elongation sufficiently before the hypothetical forces constricting the nerve trunk in silicone tubes became sufficiently large. The combined data suggest a new mechanism for peripheral nerve regeneration along a tubulated gap.

Comparison of cultured and uncultured keratinocytes seeded into a collagen–GAG matrix for skin replacements

A well-characterised collagen–glycosaminoglycan (CG) matrix functions as an extracellular matrix analogue (ECMA) of dermis on full-thickness wounds. The epidermis can be reconstituted by seeding autologous uncultured keratinocytes into the matrix prior to grafting. We hypothesised that seeding the CG matrix with keratinocytes cultured to sub-confluence may provide the ECMA with more proliferating keratinocytes than with uncultured keratinocytes. Autologous cells were isolated from split-thickness skin grafts and cultured to sub-confluence. ECMAs were seeded by centrifuging cultured (n= 8) or uncultured (n= 8) autologous keratinocytes into a CG matrix at a density of 100 000 cells/cm2, then applied onto full-thickness wounds on Yorkshire pigs. Gross and histologic observations were made up to 21 days post-grafting. At 14 days, a fully differentiated epidermis was present on all graft sites, but the epidermis of the cultured-cell-seeded matrices was thicker, 180 (19) μm, than the uncultured-cell-seeded matrices, 110 (18) μm. The epidermis of cultured-cell-seeded matrices was acanthotic, containing 14 (4) cell layers, as compared to uncultured-cell-seeded matrices, 9 (1) cell layers. The number of sub-epithelial keratinocyte cysts/cm cross-section present in the neodermis was also greater in cultured-, 1.35 (0.37), than in uncultured-cell-seeded matrices, 0.47 (0.35). Epidermal confluence on day 14 was 96 (3)% on cultured-cell-seeded grafts and 50 (17)% on uncultured-cell-seeded grafts. These results are consistent with the hypothesis that the process of in vitro cell cultivation increases the proportion of dividing cells in preference to differentiated cells. This technology may be useful in reconstruction of specialised bilayer tissues with minimal donor sites.

Early peripheral nerve healing in collagen and silicone tube implants: Myofibroblasts and the cellular response

Injuries to peripheral nerves innervating a limb cause paralysis, and can necessitate amputation. The inability of the nerves to regenerate spontaneously and the limitations of autograft procedures led to the development of treatments involving insertion of the nerve ends into prosthetic tubular devices. Previous work showed that ‘entubulation’ of the nerve ends in a silicone tube containing a specific porous, resorbable collagen-GAG (CG) copolymer, serving as an analog of extracellular matrix, improved regeneration compared to an empty silicone tube. However, long-term treatment with silicone tubes produced constriction that caused partial degradation of the regenerated axons; for this and other reasons, implementation of a nondegradable tube may require a second surgical procedure for removal. In this study the silicone tube was replaced with porous and non-porous collagen tubes in order to produce fully degradable devices. CG-filled collagen tubes and controls (CG-filled silicone tubes and empty collagen and silicone tubes) were implanted in a 10-mm gap in the rat sciatic nerve, with three rats in each group. The regeneration was evaluated after six weeks using light microscope images of cross sections of the nerve that were digitized and analyzed. Histograms of the diameters of the axons were generated and compared. The cellular response to the implanted biomaterials was assessed histologically, and immunohistochemistry was performed using an antibody to α-smooth muscle actin in order to determine the presence of myofibroblasts (contractile cells). Axonal regrowth was comparable in porous collagen, non-porous collagen, and silicone tubes filled with a CG matrix. These results support the implementation of a degradable collagen tube in place of a silicone device. Confirming earlier work, regeneration through the silicone and collagen tubes was enhanced by the CG copolymer, compared to empty tubes. A notable finding was a continuous layer of myofibroblasts on the surfaces of all of the six silicone tube prostheses, but on the inner surface of only one of six collagen tubes (Fisher’s exact tests; P<0.01). This is the first report of contractile capsules around silicone tubes, and supports the use of degradable collagen tubes in peripheral nerve regeneration. Macrophages were found bordering both the silicone and collagen tubes, and in the case of the collagen tubes, appeared to be participating in the regulation of the tubes.

Reconstructed skin from co-cultured human keratinocytes and fibroblasts on a chitosane cross-linked collagen-GAG matrix

Reconstruction of skin requires both the dermal and epidermal equivalent of the skin. A reconstructed skin has been developed, composed of two compartments: (1) a dermal equivalent (DE) comprising an acellular dermal substrate (DS) populated by foreskin fibroblasts (FF); (2) an epidermis regenerated from normal human keratinocytes (NHK) seeded on to the DE. The DS contains Types I and III collagen and glycosaminoglycans (GAGs) cross-linked by chitosane. FF seeded into the porous structure of the DS provide a DE suitable to support epidermal cells. NHK attach quickly, exhibit mitotic activity and form a continuous and stratified epidermis. After 2 weeks culture, histological sections of the RS show a basal layer with cuboidal cells attached to the DE and several suprabasal cell layers including the stratum corneum (SC). Transmission electron microscopy revealed the cell membrane densification (hemidesmosomes) at the dermal-epidermal junction; however, the lamina densa was found to be discontinuous at this stage. We noted the presence of lipid vesicles in spinous layer and keratohyalin granules in granular layer. Terminal epidermal differentiation was complete with the SC consisting of several layers of corneocytes filled with tonofilaments. Immunofluorescence studies revealed the presence of bullous pemphigoide antigen and Type IV collagen at the dermal-epidermal junction as well as the presence of keratins 1/10 and filaggrin in the suprabasal layers characteristic for epidermal differentiation. Reconstructed skin based on our chitosane cross-linked collagen-GAG matrix is morphologically equivalent to normal human skin and should thus provide a useful tool for the treatment of patients with severe burns.

Characterization of Skin Reconstructed on a Chitosan-Cross-Linked Collagen-Glycosaminoglycan Matrix

Reconstruction of skin requires both the dermal and epidermal equivalent of the skin. We have developed a reconstructed skin composed of two compartments: (1) a dermal equivalent comprising an acellular dermal substrate populated by foreskin fibroblasts and (2) an epidermis regenerated from normal human keratinocytes seeded onto the dermal equivalent. The dermal substrate contains type I and III collagen and glycosaminoglycans (GAGs) cross-linked by chitosan. Fibroblasts seeded into the porous structure of the dermal substrate provide a dermal equivalent suitable to support epidermal cells. Keratinocytes attach quickly, exhibit mitotic activity and form a continuous and stratified epidermis. After 2 weeks of culture, histological sections show a basal layer with cuboidal cells attached to the dermal equivalent and several suprabasal cell layers including the stratum corneum. Transmission electron microscopy revealed the cell membrane densification (hemidesmosomes) at the dermoepidermal junction; however, the lamina densa was found discontinuous at this stage. We noted the presence of lipid vesicles in spinous layer and keratohyalin granules in granular layer. The epidermal differentiation was complete terminal with the stratum corneum containing several layers of corneocytes filled with tonofilaments. Reconstructed skin, based on our chitosan-cross-linked collagen-GAG matrix is morphologically equivalent to normal human skin and should thus provide a useful tool for in vitro toxicological studies as well as a suitable wound covering for the treatment of patients with severe burns.