Use of the parabiotic model in studies of cutaneous wound healing to define the participation of circulating cells.

Previous experimental studies to assess the contribution of blood-borne circulating (BBC) cells to cutaneous wound healing have relied on discontinuous pulsing of labeled BBC elements or bone marrow transplant protocols. Such approaches do not allow the examination of stable BBC cells that have matured in a physiologically normal host. We have used a parabiotic murine model for cutaneous wound healing to evaluate the relative contribution of stable populations of peripheral blood cells expressing the green fluorescent protein (GFP) transgene in otherwise normal animals. Circulating cells (mature and immature) expressing the GFP transgene were easily detected and quantified in wounds of GFP- parabiotic twins during all evaluated stages of the healing response. Using multiple antibody probes, the relative contribution of various subsets of BBC cells could be comparatively assessed. In early wounds, some cells expressing mesenchymal epitopes were documented to be of hematopoietic origin, indicating the utility of this model in assessing cell plasticity in the context of tissue regeneration and repair. Application of this approach enables further investigation into the contribution of peripheral blood in normal and abnormal healing responses.

Massive internal fracture of an amorphous polyester.

When amorphious polyethylene terephthalate is subjected to a tensile load of 2 to 4 x 10(8) dynes per square centimeter wthin the approximate temperature range 40 degrees to 70 degrees C, an unusual optical effect occurs. The transparent polymer film is suddenly transformed into a brilliantly reflecting strip with the luster of silver. Extensive formation of voids accounts for the unorthodox behavior.

Wound tissue can utilize a polymeric template to synthesize a functional extension of skin.

Prompt and long-term closure of full-thickness skin wounds is guinea pigs and humans is achieved by applying a bilayer polymeric membrane. The membrane comprises a top layer of a silicone elastomer and a bottom layer of a porous cross-linked network of collagen and glycosaminoglycan. The bottom layer can be seeded with a small number of autologous basal cells before grafting. No immunosuppression is used and infection, exudation, and rejection are absent. Host tissue utilizes the sterile membrane as a culture medium to synthesize neoepidermal and neodermal tissue. A functional extension of skin over the entire wound area is formed in about 4 weeks.

Raman spectroscopy: a structural probe of glycosaminoglycans.

We report the first Raman spectroscopic study of the glycosaminoglycans chondroitin 4-sulfate, chondroitin 6-sulfate and hyaluronic acid, both in solution and in the solid state. To aid in spectral identification, infrared spectra were also recorded from films of these samples. Vibrational frequencies for important functional groups like the sulfate groups, glycosidic linkages, C-OH and the N-acetyl group can be identified from the Raman spectra. Certain differences in the spectra of the different glycosaminoglycans can be interpreted in terms of the geometry of the various substituents, while other differences can be related to differences in chemical composition.

The effect of pore size on cell adhesion in collagen-GAG scaffolds.

The biological activity of scaffolds used in tissue engineering applications hypothetically depends on the density of available ligands, scaffold sites at which specific cell binding occurs. Ligand density is characterized by the composition of the scaffold, which defines the surface density of ligands, and by the specific surface area of the scaffold, which defines the total surface of the structure exposed to the cells. It has been previously shown that collagen-glycosaminoglycan (CG) scaffolds used for studies of skin regeneration were inactive when the mean pore size was either lower than 20 microm or higher than 120 microm (Proc. Natl. Acad. Sci., USA 86(3) (1989) 933). To study the relationship between cell attachment and viability in scaffolds and the scaffold structure, CG scaffolds with a constant composition and solid volume fraction (0.005), but with four different pore sizes corresponding to four levels of specific surface area were manufactured using a lyophilization technique. MC3T3-E1 mouse clonal osteogenic cells were seeded onto the four scaffold types and maintained in culture. At the experimental end point (24 or 48 h), the remaining viable cells were counted to determine the percent cell attachment. A significant difference in viable cell attachment was observed in scaffolds with different mean pore sizes after 24 and 48 h; however, there was no significant change in cell attachment between 24 and 48 h for any group. The fraction of viable cells attached to the CG scaffold decreased with increasing mean pore size, increasing linearly (R2 = 0.95, 0.91 at 24 and 48 h, respectively) with the specific surface area of the scaffold. The strong correlation between the scaffold specific surface area and cell attachment indicates that cell attachment and viability are primarily influenced by scaffold specific surface area over this range (95.9-150.5 microm) of pore sizes for MC3T3 cells.

Surface biology of collagen scaffold explains blocking of wound contraction and regeneration of skin and peripheral nerves.

We review the details of preparation and of the recently elucidated mechanism of biological (regenerative) activity of a collagen scaffold (dermis regeneration template, DRT) that has induced regeneration of skin and peripheral nerves (PN) in a variety of animal models and in the clinic. DRT is a 3D protein network with optimized pore size in the range 20-125 µm, degradation half-life 14 ± 7 d and ligand densities that exceed 200 µM α1ß1 or α2ß1 ligands. The pore has been optimized to allow migration of contractile cells (myofibroblasts, MFB) into the scaffold and to provide sufficient specific surface for cell-scaffold interaction; the degradation half-life provides the required time window for satisfactory binding interaction of MFB with the scaffold surface; and the ligand density supplies the appropriate ligands for specific binding of MFB on the scaffold surface. A dramatic change in MFB phenotype takes place following MFB-scaffold binding which has been shown to result in blocking of wound contraction. In both skin wounds and PN wounds the evidence has shown clearly that contraction blocking by DRT is followed by induction of regeneration of nearly perfect organs. The biologically active structure of DRT is required for contraction blocking; well-matched collagen scaffold controls of DRT, with structures that varied from that of DRT, have failed to induce regeneration. Careful processing of collagen scaffolds is required for adequate biological activity of the scaffold surface. The newly understood mechanism provides a relatively complete paradigm of regenerative medicine that can be used to prepare scaffolds that may induce regeneration of other organs in future studies.

Delivery of plasmid DNA to articular chondrocytes via novel collagen-glycosaminoglycan matrices.

Our primary objective was to fabricate a porous gene-supplemented collagen-glycosaminoglycan (GSCG) matrix for sustained delivery (over a period of several weeks) of plasmid DNA to articular chondrocytes when implanted into cartilage lesions. The specific aims of this in vitro study were to determine the release kinetics profiles of plasmid DNA from the GSCG matrices, and to determine the ability of the released plasmid DNA to transfect adult canine articular chondrocytes. In particular, we evaluated the effects of two variables, cross-linking treatment and the pH at which the DNA was incorporated into the matrices, on the amount of the plasmid DNA that remained bound to the GSCG matrices after passive (nonenzymatic) leaching and on the expression of a reporter gene in articular chondrocytes grown in the GSCG matrices. Collagen-glycosaminoglycan matrices were synthesized without cross-linking, and by three cross-linking treatments: dehydrothermal (DHT) treatment, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) treatment, and exposure to ultraviolet (UV) radiation. The plasmid DNA was incorporated into the collagen-glycosaminoglycan matrices in solutions at pH 2.5 or 7.5. Transmission electron microscopy studies revealed plasmid DNA bound to the walls of the porous GSCG matrices. In general, the GSCG matrices fabricated at pH 2.5 retained a larger fraction of the initial DNA load after 28 days of incubation in Tris-EDTA buffer. The passive, solvent-mediated release of the plasmid DNA from the GSCG matrices showed a biphasic pattern consisting of a faster, early release rate over the initial 8 hr of leaching followed by a slower, late release rate that was relatively constant over the subsequent 28 days of leaching. Electrophoretic analyses revealed that the plasmid DNA released from the GSCG matrices fabricated at pH 2.5 had been linearized and/or degraded; whereas the plasmid DNA leached from the GSCG matrices prepared with a DNA solution at pH 7.5 was primarily supercoiled and linear. Plasmid DNA released from all GSCG matrix formulations was able to generate luciferase reporter gene expression in monolayer-cultured chondrocytes transfected with the aid of a commercial lipid reagent, and in chondrocytes cultured in the GSCG matrices without the aid of a supplemental transfection reagent. Luciferase expression in chondrocyte-seeded GSCG constructs was evident throughout the culture period (28 days), with the EDC and UV cross-linked matrices prepared at pH 7.5 providing the highest transgene expression levels. We conclude that released plasmid DNA continually transfected canine articular chondrocytes seeded into GSCG matrices in vitro for a 4-week period as evidenced by luciferase reporter gene expression. Thus, GSCG matrices can be fabricated to provide sustained release of plasmid DNA carrying a potential therapeutic gene.

Scattering of light from histologic sections: a new method for the analysis of connective tissue.

We have developed a light scattering technique that can be used to analyze the orientation and diameter of collagen fibers in histologic sections of connective tissue. Scattering patterns obtained by transmitting laser light through sections of tissue contain information both on the orientation, degree of alignment, and size of the constituent collagen fibers. Analysis of the azimuthal intensity distribution of scattered light yields numerical values of the degree of alignment by use of an orientation index, S, which is chosen to vary between 0 for randomly oriented fibers and 1 for a perfectly aligned arrangement. The average diameter of the collagen fibers is calculated from the scattering angle at which the intensity reaches its first minimum. These measurements are independent of the nature of histologic stain. The procedure is illustrated by measurements obtained with sections of the guinea pig dermis and of control scar. We conclude from our experiments that light scattering can complement the analysis of tissue architecture typically performed with the light microscope.

Organized skin structure is regenerated in vivo from collagen-GAG matrices seeded with autologous keratinocytes.

A well-characterized collagen-glycosaminoglycan matrix (CGM) that has been shown to function as a dermal analog was seeded with freshly disaggregated autologous keratinocytes and applied to full-thickness wounds in a porcine model. CGM were impregnated with 50,000 keratinocytes per cm2, a seeding density that produces a confluent epidermis within 19 d post-grafting and affords a 60-fold surface expansion of the donor epidermis. In this study, the temporal sequence of events in epidermal and neodermal formation was analyzed histopathologically and immunohistochemically from 4 to 35 d post-grafting. The epidermis was observed to form from clonal growth of individual keratinocytes into epithelial cords and islands that gradually enlarged, coalesced, differentiated to form large horn cysts, and finally reorganized at the graft surface to form a fully differentiated, normally oriented epidermis with rete ridges. Simultaneously, a neodermis formed from migration of endothelial cells, fibroblasts, and macrophages into the CGM from the underlying wound bed, resulting in formation of blood vessels, the production of abundant extracellular matrix, and the degradation of the CGM fibers, respectively. Gradually, the stromal cellularity of the CGM decreased and collagen deposition and remodeling increased to form a neodermal connective tissue matrix beneath the newly formed epidermis. Complete dissolution of the CGM occurred, partly as a result of degradation by an ongoing foreign-body giant cell reaction that peaked at 8-12 d post-grafting, but neither acute inflammation nor evidence of immune stimulation were observed. Within 1 mo, many structural components of normal skin were reconstituted.

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.

Inhibition of conjunctival scarring and contraction by a porous collagen-glycosaminoglycan implant.

PURPOSE: To study the healing processes of full-thickness wounds in the adult rabbit conjunctiva after grafting with a porous collagen-glycosaminoglycan (CG) copolymer matrix. METHODS: A 7-mm trephine was used to produce lesions of the bulbar conjunctiva down to the level of the bare sclera. Full-thickness removal of the conjunctiva and Tenon's capsule created a reproducible wound bed. Wounds either remained ungrafted (control) or were grafted with CG matrix. In previous studies, this CG matrix has induced partial regeneration of the dermis in the human, the swine, and the guinea pig. Healing of the conjunctival epithelium and underlying stroma was evaluated by histology, immunohistochemistry, and measurement of wound contraction kinetics. RESULTS: By 28 days, ungrafted wounds had closed by contraction (26.4% +/- 5.0% fornix shortening) and the formation of scarlike tissue comprising an aligned array of dense collagen populated with occasional fibroblasts. Grafting of identical defects with CG copolymer matrix resulted in inhibition of wound contraction (6.8% +/- 3.2% fornix shortening) and the formation of a tissue that resembled normal conjunctival stroma, being composed of a loose network of collagen fibers and fibroblasts. Contractile fibroblasts (myofibroblasts) were identified at the edge of both ungrafted and grafted wounds during the period of active contraction. Both ungrafted and grafted wounds were completely re-epithelialized by 28 days. CONCLUSIONS: Implantation of CG copolymer matrix drastically reduced contraction and promoted the formation of a nearly normal subconjunctival stroma.

Recent advances in tissue synthesis in vivo by use of collagen-glycosaminoglycan copolymers.

Biologically active analogues of the extracellular matrix (ECM) are synthesized by grafting glycosaminoglycan (GAG) chains onto type I collagen, and by controlling the physicochemical properties of the resulting graft copolymer. Collagen-GAG ECM analogues have previously been shown to induce regeneration of the dermis in humans and the guinea pig, and of the rat sciatic nerve. Current studies have emphasized elucidation of the molecular mechanism through which tissue-specific ECM analogues induce regeneration. The contribution of the GAGs to the biological activity of the skin regeneration template was confirmed by studying the contribution of several GAGs to the inhibition of wound contraction in guinea pigs. The interaction between cells and the porous structure of an ECM analogue was studied with emphasis on the deformation of pores which occurs during wound contraction. The synthesis of scar, as well as of partly regenerated tissue which has a morphology between that appropriate for scar and for normal dermis, was quantitatively assayed for the first time using a laser light scattering technique. An ECM analogue which has been shown to be capable of inducing regeneration of functional sciatic nerve in the rat over a gap larger than 10 mm was incorporated in the design of a biodegradable implant for peripheral nerve regeneration.

Pore strain behaviour of collagen-glycosaminoglycan analogues of extracellular matrix.

Mechanical properties of a highly porous collagen-glycosaminoglycan (CG) analogue of extracellular matrix were investigated. The stress-strain behaviour of the matrices displayed the typical concave-up shape characteristic of porous materials and soft tissues. Deformation behaviour of the material was further investigated using novel methodology to determine material strains at the micrometre level. Central to the methodology was the use of scanning electron microscopy (SEM) in conjunction with a digital image analyser. The method for strain determination at the micrometre level was verified by comparison with measurement of surface strain using an optical method. An unexpected finding in these materials is that Poisson's ratio varies with engineering strain. A geometrical/mathematical model was developed to explain this behaviour. These results encourage further investigation of the mechanical properties of these materials, in order to provide important insights into the physical microenvironment in which cells reside.

Tendon cell contraction of collagen-GAG matrices in vitro: effect of cross-linking.

The contraction of connective tissue cells can play important roles in wound healing and pathological contractures. The effects of this contractile behavior on cell-seeded constructs for tissue engineering have not yet been investigated. The goal of this work was to investigate in vitro tendon cell-mediated contraction of collagen-glycosaminoglycan (GAG) matrices cross-linked using selected methods. Highly porous collagen-GAG sponges were seeded with calf tendon cells and the projected area and DNA content of the sponges measured at 3, 7, 14, and 21 days post-seeding. Immunohistochemistry was performed to determine if alpha-smooth muscle actin (SMA) was associated with the cell contraction of the matrices. Dehydrothermal (DHT) treatment alone was not sufficient to resist contraction by the seeded tendon fibroblasts. Cross-linking of the collagen-GAG sponges to the extent that the modulus was three times that of sponges treated by DHT alone was necessary to resist contraction. SMA was seen in the cytoplasm of most cells in all sponges at all time periods. The results provide a rational basis for the determination of the mechanical properties of collagen matrices required for engineering certain connective tissues.

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 microm 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.

Contraction of collagen-glycosaminoglycan matrices by peripheral nerve cells in vitro.

The objective of this study was to investigate the contractile behavior of peripheral nerve support cells in collagen-glycosaminoglycan (GAG) matrices in vitro. Contractile fibroblasts (myofibroblasts) are known to participate in wound contraction during healing of selected connective tissues (viz., dermis), but little is known about the activity of non-muscle contractile cells during healing of peripheral nerves. Explants from adult rat sciatic nerves were placed onto collagen-GAG matrix disks and maintained in culture for up to 30 days. Groups of collagen-GAG matrices were tested that differed in average pore diameter and in degree of cross-linking. Cell migration from nerve explants into the matrices was examined, and immunohistochemical staining was used to identify cells expressing a contractile actin isoform (alpha-smooth muscle actin; alpha-SMA) and Schwann cells (S-100). Geometric contraction of matrix disks was quantified every five days as the percent reduction in disk diameter. The amount of contraction of matrix disks was significantly affected by the degree of cross-linking. Cell migration into the matrices and the distribution of cells staining for alpha-SMA or S-100 was not affected by matrix parameters. These studies demonstrate that cells from peripheral nerve explants were capable of adopting a contractile phenotype and causing geometric contraction of matrices in vitro and suggest that contractile processes may be important during nerve wound healing in vivo.

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.

Meniscus cells seeded in type I and type II collagen-GAG matrices in vitro.

The objective of this study was to determine the proliferative and biosynthetic activity of calf meniscus cells seeded in type I and type II collagen-glycosaminoglycan (GAG) copolymers with the overall goal to develop a cell-seeded implant for future investigations to improve the regeneration of the knee meniscus. The cell-seeded matrices were digested in protease and analyzed for GAG by a modification of the dimethyl-methylene blue method and assayed for DNA content. Other specimens were evaluated histologically after 1, 7, 14 and 21 days. Contraction of the same types of matrices, seeded with adult canine meniscus cells, was measured at the same time points. After three weeks, cells were observed throughout the type II matrix, whereas the type I matrix was densely populated at the margins. The cell morphology and the cell density after three weeks in both matrices was consistent with the normal meniscus. DNA assay for the type I matrix showed a 40% decrease over the first week and a final amount of DNA that was not significantly different from the initial value, whereas the type II matrix doubled its DNA content over the same time period. The cells continued their biosynthesis of GAG and type I collagen. GAG content of the type II matrix increased by 50% more than the type I matrix after three weeks. Over the same time period, the type I matrix displayed a significant shrinkage to approximately 50% of its initial value whereas in contrast, the type II matrix and the unseeded controls showed no significant shrinkage. The number of cells and the higher GAG synthesis in the type II matrix, and its resistance to cell-mediated contracture, commend it for future investigation of the regeneration of meniscus in vivo.

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 alpha-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.

Matrix collagen type and pore size influence behaviour of seeded canine chondrocytes.

This study directly compared the behaviour of chondrocytes in porous matrices comprising different collagen types and different pore diameters. There was a dramatic difference in the morphology of the cells in the type I and type II collagen matrices. The cells in the type II collagen matrix retained their chondrocytic morphology and synthesized glycosaminoglycans, while in the type I matrix the chondrocytes displayed a fibroblastic morphology with less biosynthetic activity than those in the type II. Small pore diameter affected morphology initially in the type I matrices and showed a higher increase of DNA content, but with time the cells lost the chondrocytic morphology. Our results demonstrate the marked influence of collagen type and pore characteristics on the phenotypic expression of seeded chondrocytes.