Diffusivity of 125I-labelled macromolecules through collagen: mechanism of diffusion and effect of adsorption

Diffusion of angiotensin II, albumin and aldolase was studied through collagen membranes with swelling ratios between 4 and 15. The diffusion coefficient was measured from the time-lag for the onset of steady-state flux through the membrane. Binding of macromolecules to collagen was evaluated from the results of sorption studies conducted as a function of macromolecular concentration. Results presented indicate that the diffusion of macromolecules through collagen membrane is slowed by electrostatic and hydrogen bonding between individual macromolecular chains and collagen. The extent of adsorption is increased as the molecular weight of the diffusant increases. Diffusion of water soluble macromolecules through collagen occurs rapidly, suggesting that diffusion occurs through water filled channels as opposed to between collagen molecules. The results of these studies are useful in understanding diffusion through connective tissues and in the design of drug delivery systems based on collagen.     

Control of vitamin B12 release from poly(ethylene glycol)/poly(butylene terephthalate) multiblock copolymers

The release of vitamin B12 (1355 Da) from matrices based on multiblock copolymers was studied. The copolymers were composed of hydrophilic poly(ethylene glycol)-terephthalate (PEGT) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks. Vitamin B12 loaded films were prepared by using a water-in-oil emulsion method. The copolymer properties, like permeability, could be varied by increasing the PEG-segment length from 300 up to 4,000 g/mol and by changing the wt% of PEGT. From permeation and release experiments. the diffusion coefficient of vitamin B12 through PEGT/PBT films of different compositions was determined. The diffusion coefficient of Vitamin B12 was strongly dependent on the composition of the copolymers. Although an increased wt% of PEGT (at a constant PEG-segment length) resulted in a higher diffusion coefficient, a major effect was observed at increasing PEG-segment length. By varying the copolymer composition, a complete release of vitamin B12 in 1 day up to a constant release for over 12 weeks was obtained. The release rate could be effectively tailored by blending copolymers with different PEG-segment lengths. The swelling and the crystallinity of the matrix could explain the effect of the matrix composition on the release behavior.     

The impact of proteinase-induced matrix degradation on the release of VEGF from heparinized collagen matrices

The in vivo application of engineered matrices in human wound healing processes is often hampered by the slow rate of vascularization. Therefore much research is directed towards enhancing the angiogenic properties of such matrices. One approach for enhancing the vascularization is the incorporation of angiogenic growth factors. Recently, we and others have reported on immobilizing such factors into collagen matrices either by covalent attachment or by physical binding to covalently incorporated heparin. Especially the latter procedure has been shown to lead to substantial increase rates in vascularization in in vivo experiments. The increases have been proposed to depend on the sustained release of the incorporated angiogenic growth factors from the heparinized collagen matrices. In this paper, we report on investigations to study the release of vascular endothelial growth factor (VEGF) from collagen matrices under conditions which mimic potential in vivo situations. Relevant proteinase concentrations were deduced from in vitro experiments in which we evaluated the secretion of selected matrix metalloproteinases from fibroblasts in contact with collagen. The release of VEGF from non-modified, cross-linked and heparinized collagen matrices in the absence and in the presence of varying concentrations of proteinases was then determined by ELISA and liquid scintillation counting. The release behaviour appears to be controlled by both the presence of heparin and the levels of proteinases applied. Experiments with matrices containing radioactively labelled heparin suggest that VEGF release results from the consecutive and simultaneous release of three species of VEGF molecules that differ in their binding affinities to the differently modified collagen matrices. The species binding specifically to heparin most likely accounts for the previously observed increases in angiogenic potential between loading VEGF to non-heparinized and heparinized collagen matrices.     

Unexpected observation of concentration-dependent dissociation rates for antibody-antigen complexes and other macromolecular complexes in competition experiments

The dissociation rate constant of bimolecular complexes between macromolecules (k(off)) is often measured in solution by competition experiments and is generally expected to follow first-order kinetics.When measuring k(off) constants by competition for three complexes of high-affinity recombinant antibody fragments with the cognate antigen and for one calmodulin/peptide complex, a surprising dependence between apparent dissociation rate and concentration of competitor (antigen or calmodulin-binding peptide) was observed.Our results may be characteristic for macromolecules consisting of two domains (such as single-chain Fv fragments) and may reflect a transient opening of the two domains which are involved in the binding reaction, and which are connected by a polypeptide linker.     

Impact of cell type and density on nerve growth factor distribution and bioactivity in 3-dimensional collagen gel cultures

Local delivery of protein agents is potentially important in many tissue engineering systems. In this report, we evaluate an experimental system for measuring the rate of nerve growth factor (NGF) transport and biological activity within a 3-dimensional, tissue-like environment. Fetal brain cells or PC12 cells were suspended throughout collagen gel cultures; controlled-release matrices were used to control the spatial and temporal pattern of NGF release. Experimentally measured concentration profiles were compared to profiles predicted by a mathematical model encompassing diffusion and first-order elimination. Our results suggest that NGF moves through gels by diffusion while being eliminated at a rate that depends on cell density. Since diffusion and elimination also govern protein transport in brain tissue, the collagen gel serves as a model system that replicates the main features of transport in the brain and, therefore, can be used to identify new strategies that enhance NGF distribution in the central nervous system. As an example of the utility of this biophysical model, we demonstrate that implantation of multiple controlled-release matrices can broaden NGF distribution in gel cultures; this broadening was accompanied by a significant increase in cellular biological activity. This approach may be useful in customizing NGF distribution throughout degenerating or damaged central nervous system tissue while minimizing toxicity to surrounding healthy tissue.     

The effect of cross-linking of collagen matrices on their angiogenic capability

The poor vascularization rate of matrices following cell invasion is considered to be one of the main shortcomings of scaffolds used in tissue engineering. In the past decade much effort has been directed towards enhancing the angiogenic potential of biomaterials. A great many studies have appeared reporting about enhancement of vascularization by immobilizing angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-2 (FGF-2). We have also tried to achieve this goal by modifying collagen matrices by covalent incorporation of heparin into the matrices and loading them with VEGF. We and others have observed that loading angiogenic factors to heparinized materials markedly increases angiogenic capacity. In the present paper we also investigated the angiogenic properties of collagen matrices which were only cross-linked, i.e. in the absence of heparin. The angiogenic capacity of the modified matrices was evaluated using the chorioallantoic membrane assay. Differences in angiogenic potential were deduced from macroscopic and microscopic analyses of the chorioallantoic membrane, as well as from dry weight changes. Cross-linked only matrices and matrices both cross-linked and heparinized appeared to show a significantly larger angiogenic potential than unmodified matrices. As previously observed, loading VEGF to these matrices further stepped up angiogenic potential. Quite surprisingly, cross-linking had a substantial impact on angiogenic potential. In terms of magnitude, this effect was similar to the effect of loading VEGF to heparinized matrices. Both modification procedures resulted in an increase of average pore size within the collagen matrices, and this observation may explain the more rapid invasion of mouse fibroblasts into cross-linked and heparinized matrices. Form changes of the implants were also monitored during the in vivo contacts: cross-linked and heparinized matrices showed far better resistance against contraction, as compared to unmodified matrices. Results from the chorioallantoic membrane assay experiments were compared with data obtained from rat model experiments, which confirmed the results from the chorioallantoic membrane assay. This relatively simple assay was again shown to be extremely helpful in evaluating and predicting the angiogenic capabilities of biomaterials for use in tissue engineering and wound healing.     

Biodegradation of carbonate apatite/collagen composite membrane and its controlled release of carbonate apatite

The aim of this study was to investigate the biodegradation of carbonate apatite (CO(3)Ap)/collagen composite membrane as a new guided tissue regeneration membrane in vivo and to estimate its controlled release of CO(3)Ap in vitro. To control the biodegradation of the guided tissue regeneration membrane and to promote hard tissue regeneration in the periodontal region, we added CO(3)Ap into the collagen membrane. To investigate the biodegradation of CO(3)Ap/collagen composite membranes, the prepared membranes (CO(3)Ap:0, 10 wt %) were cut into 5 x 5 x 0.1 mm and subcutaneously implanted into the backside of male rats under general anesthesia. The explanted membranes were investigated histologically. To estimate their controlled release of CO(3)Ap in vitro, the membranes (CO(3)Ap 0-10 wt %, 5 x 5 x 0.1 mm) were immersed into collagenase solution and compulsorily dissolved for 48 h. Histological results suggested that the membrane had a good biocompatibility and the biodegradable period was shortened with the presence of CO(3)Ap. In the solubility experiments of the membrane, eluted Ca concentrations gradually increased with total dependence on the dissolution of the collagen membrane. Our study demonstrated that the biodegradation time can be controlled by CO(3)Ap contents in the membrane and CO(3)Ap could be released from the membrane with the biodegradation period.     

Rational Design of Contact Guiding,Neurotrophic Matrices for Peripheral Nerve Regeneration

Nerve guides filled with magnetically aligned hydrated gels of type I collagen have been shown to impart strong contact guidance cues to elongating neurites in vitro ¹⁵ and to increase the number of regenerating axons in vivo ⁸ relative to an isotropic collagen gel. We have formulated and analyzed a model to determine the conditions under which the target concentration of nerve growth factor (NGF) to support axonal growth can be sustained by entrapping either NGF-secreting cells or NGF-releasing polymer microspheres in the aligned gel. The equation describing NGF concentration with a distributed source term was solved after experimental determination of (1) the rate of NGF release from PLGA 85/15 microspheres, (2) the NGF diffusion coefficient in the gel and nerve guide membrane containing the gel, and (3) the maximum microsphere loading that does not compromise the magnetic alignment of collagen fibrils. We find that for a rat sciatic nerve, when using a 1 mm diameter nerve guide within a commercially available collagen membrane, the microsphere loading limit will prevent the construct's capacity to sustain the target NGF concentration of 1 ng/ml at two months when either wild type Schwann cells or PLGA 85/15 microspheres are used as the NGF source. This target concentration, however, will be maintained when transfected cells described in the literature to hypersecrete NGF are used, or when the microspheres are used if the permeability of the nerve guide membrane can be moderately decreased. For a human median nerve, when using a 5 mm diameter nerve guide within a commercially available membrane, the microspheres are capable of sustaining NGF concentrations above 1 ng/ml to at least 75 days without the need to decrease membrane permeability. © 2003 Biomedical Engineering Society. PAC2003: 8780Xa, 8718Sn, 8715Vv     

Cross-linking of amniotic membranes

Human amniotic membrane was cross-linked with chemical and radiation methods to investigate the effect of cross-linking on its physicochemical and biodegradation properties. Radiation cross-linking was performed with gamma-ray and electron beam while chemical cross-linking was with glutaraldehyde (GA). Both gamma-ray and electron beam irradiation decreased the tensile strength and elongation at break of the amniotic membrane with an increase in the irradiation dose, whereas GA cross-linking had no effect on the tensile properties. This is probably due to the scission of collagen chains through irradiation. No significant change was observed on the water content of cross-linked amniotic membranes for any of the crosslinking methods and in marked contrast with cross-linking of a gelatin membrane. A permeation study revealed that protein permeation through the amniotic membrane was not influenced by the GA concentration at cross-linking. These findings are ascribed to the structure characteristic of the amniotic membrane. The membrane is composed of a fibrous mesh structure from an assemblage of collagen fibers. It is possible that cross-linking takes place in the interior of the fiber assembly without impairing the mesh structure, resulting in no change of the water content and protein permeability. In vitro degradation of cross-linked amniotic membranes revealed that radiation cross-linking appeared to be much less effective than GA cross-linking in retarding the degradation, probably because of low cross-linking densities. GA-cross-linked amniotic membranes were degraded more slowly as the GA concentration at cross-linking increased. When the GA-cross-linked amniotic membrane was subcutaneously implanted in the rat, the tissue response was mild, similar to that of the non-cross-linked native membrane.     

A highly flexible paclitaxel-loaded poly(ε-caprolactone) electrospun fibrous-membrane-covered stent for benign cardia stricture

In benign esophageal strictures, inflammation reaction and tissue hyperplasia after stent placement greatly limit the stent retention time and affect subsequent scar formation, which is one of the main influences on long-term recurrence rate. A newly developed biodegradable electrospun drug-fiber-coated stent (DFCS) was fabricated to inhibit inflammation and scar formation. The electrospun paclitaxel/poly(ε-caprolactone) (PCL) fibers integrally covered the bare stent using the rotating collection method. The paclitaxel entrapment did not significantly affect the physical properties of electrospun PCL fibrous membranes. The mechanical results demonstrated that electrospun fibers containing paclitaxel covering the stent maintained the original mechanical characteristics of the stent, and no membrane tearing or ablation was observed after hundreds of repeated compressions. Paclitaxel release profiles were controlled mainly via diffusion of drug through the drug content, and stable release of paclitaxel continued up to 32 days at pH 4.0. Higher inhibition of smooth muscle cell proliferation rates was observed on fibrous membranes with higher paclitaxel content. DFCS showed a significant decrease in tissue inflammation and collagen fiber proliferation, and was easily removed from the esophageal part, which had almost no damage to the tissues in the dog model. Therefore, DFCSs may have great potential to markedly attenuate stent-induced inflammation and scar formation in esophageal stenosis therapy.     

Macromolecules released from polymers: diffusion into unstirred fluids

Polymers that release macromolecules may be useful for preventing and treating human disease. In certain applications of polymeric controlled release, like drug therapy of brain disease and immunoprotection of mucus epithelia, effectiveness may be limited by diffusion through an unstirred fluid near the polymer. Using computer-assisted epifluorescence microscopy, we have examined the local distribution of fluorescently labelled macromolecules released from an ethylene-vinyl acetate copolymer matrix into unstirred layers of phosphate-buffered water and mid-cycle human cervical mucus. Diffusion coefficients in the fluid were determined by observing the concentration profiles as a function of time. Diffusion coefficients determined for fluorescein, bovine serum albumin, and three classes of human immunoglobulins (IgG, slgA and IgM) in phosphate-buffered water were in good agreement with literature values. For fluorescein, albumin and IgG, diffusion in mucus was comparable with diffusion in water: the largest molecule tested was slowed by only a factor of 3.     

Cross-linking of amniotic membranes

Abstraet-Human amniotic membrane was cross-linked with chemical and radiation methods to investigate the effect of cross-linking on its physicochemical and biodegradation properties. Radiation cross-linking was performed with γ-ray and electron beam while chemical cross-linking was with glutaraldehyde (GA). Both γ-ray and electron beam irradiation decreased the tensile strength and elongation at break of the amniotic membrane with an increase in the irradiation dose, whereas GA cross-linking had no effect on the tensile properties. This is probably due to the scission of collagen chains through irradiation. No significant change was observed on the water content of cross-linked amniotic membranes for any of the crosslinking methods and in marked contrast with cross-linking of a gelatin membrane. A permeation study revealed that protein permeation through the amniotic membrane was not influenced by the GA concentration at cross-linking. These findings are ascribed to the structure characteristic of the amniotic membrane. The membrane is composed of a fibrous mesh structure from an assemblage of collagen fibers. It is possible that cross-linking takes place in the interior of the fiber assembly without impairing the mesh structure, resulting in no change of the water content and protein permeability. In vitro degradation of cross-linked amniotic membranes revealed that radiation cross-linking appeared to be much less effective than GA cross-linking in retarding the degradation, probably because of low cross-linking densities. GA-cross-linked amniotic membranes were degraded more slowly as the GA concentration at cross-linking increased. When the GA-cross-linked amniotic membrane was subcutaneously implanted in the rat, the tissue response was mild, similar to that of the non-cross-linked native membrane.     

Non-cross-linked porcine-based collagen I-III membranes do not require high vascularization rates for their integration within the implantation bed: a paradigm shift

There are conflicting reports concerning the tissue reaction of small animals to porcine-based, non-cross-linked collagen I-III membranes/matrices for use in guided tissue/bone regeneration. The fast degradation of these membranes/matrices combined with transmembrane vascularization within 4 weeks has been observed in rats compared with the slow vascularization and continuous integration observed in mice. The aim of the present study was to analyze the tissue reaction to a porcine-based non-cross-linked collagen I-III membrane in mice. Using a subcutaneous implantation model, the membrane was implanted subcutaneously in mice for up to 60 days. The extent of scaffold vascularization, tissue integration and scaffold thickness were assessed using general and specialized histological methods, together with a unique histomorphometrical analysis technique. A dense Bombyx mori-derived silk fibroin membrane was used as a positive control, whilst a polytetrafluoroethylene (PTFE) membrane served as a negative control. Within the observation period, the collagen membrane induced a mononuclear cellular tissue response, including anti-inflammatory macrophages and the absence of multinucleated giant cells within its implantation bed. Transmembrane scaffold vascularization was not observed, whereas a mild scaffold vascularization was generated through microvessels located at both scaffold surfaces. However, the silk fibroin induced a mononuclear and multinucleated cell-based tissue response, in which pro-inflammatory macrophages and multinucleated giant cells were associated with an increasing transmembrane scaffold vascularization and a breakdown of the membrane within the experimental period. The PTFE membrane remained as a stable barrier throughout the study, and visible cellular degradation was not observed. However, multinucleated giant cells were located on both interfaces. The present study demonstrated that the tested non-cross-linked collagen membrane remained as a stable barrier membrane throughout the study period. The membrane integrated into the subcutaneous connective tissue and exhibited only a mild peripheral vascularization without experiencing breakdown. The silk fibroin, in contrast, induced granulation tissue formation, which resulted in its high vascularization and the breakdown of the material over time. The presence of multinucleated giant cells at both interfaces of the PFTE membrane is a sign of its slow cellular biodegradation and might lead to adhesions between the membrane and its surrounding tissue. This hypothesis could explain the observed clinical complications associated with the retrieval of these materials after guided tissue regeneration.     

Dense fibrillar collagen matrices: a model to study myofibroblast behaviour during wound healing

Fibroblastic cells play an important part in wound healing. Human dermal fibroblasts seeded onto three-dimensional fibrillar collagen matrices migrate into the collagen network and differentiate into myofibroblasts. In order to evaluate the use of collagen matrices as model systems for studying myofibroblast phenotype during wound healing, myofibroblast behaviour migrating into dense or loose matrices was compared. The effect of collagen concentration on cell morphology, remodelling, proliferation and apoptosis of human myofibroblasts was evaluated. Myofibroblasts within dense collagen matrices (40 mg/ml) were spindle shaped, similar to cells observed during tissue repair. In contrast, cells within loose matrices (5mg/ml) were more rounded. Matrix hydrolysis activities (MT1-MMP and MMP2) did not differ between the two collagen concentrations. The myofibroblast proliferation rate was measured after 24h bromodeoxyuridine incorporation (BrdU). Cells in dense collagen matrices proliferated at a higher rate than cells in loose matrices at each culture time point tested. For example, 40% of cells in dense matrices were replicating compared to 10% of cells in loose matrices after 28 days in culture. Apoptotic cells were only detected in dense matrices from day 21 onwards when cells had already migrated into the collagen network. Taken together, these results show that a high collagen concentration has a stimulatory effect on myofibroblast proliferation and apoptosis, two important events in wound healing. Thus, dense matrices can be used to create controlled conditions to study myofibroblast phenotype.     

Influence of betacyclodextrin on the release of poorly soluble drugs from inert and hydrophilic heterogeneous polymeric matrices

The release behavior of poorly soluble drugs (naproxen and ketoprofen) from inert (acrylic resins) and hydrophilic swellable (high-viscosity hydroxypropylmethylcellulose) tableted matrices containing betacyclodextrin (betaCD) was investigated. The results demonstrated that, in both cases, betaCD can enhance the rate of drug release. Matrices obtained from formulations in which lactose replaced betaCD were also evaluated. BetaCD in inert matrices causes a dramatic increase in the rate of drug release, higher than that promoted by lactose which merely acts as a channelling agent. This result suggests that possible in situ formation of the drug-betaCD complex. which causes an improvement in apparent drug solubility, could have a greater influence on the rate of drug release than the possible increase of water uptake by a soluble filler. Indeed, if the opposite were true, lactose would be more effective in increasing the rate of drug release than betaCD, because of its greater solubility in water. On the contrary, in the case of hydrophilic matrices, lactose proves to be much more effective in promoting drug release than betaCD. It seems that, while the bulky interaction compound can freely diffuse through water-filled pores of inert systems, its diffusion through swollen macromolecular chains of hydrophilic matrices may be hindered. This hypothesis was supported by data obtained from binary (drug/polymer) and ternary (drug/polymer/betaCD) hydrophilic matrices using a betaCD-containing dissolution media.     

Rationalization and optimization of hemodialysis procedure—III

For single-pass counterflow dialysers, an equation has been derived which incorporates flow rates in blood and dialysate compartments, blood volume, overall diffusion coefficient of the substance involved and geometrical membrane properties. To verify the theory effects of flow in the blood and dialysate compartments, on mass transfer of sodium through the membranes in a Kiil dialyser were studied inin vitro experiments, using water/water and blood/dialysate systems. The geometrical properties of the membranes and the overall diffusion co-efficient of sodium were determined. The latter was less in the blood/dialysate system than in the water/water system. The efficiency of this dialyser was expressed as a clearance. The flow dependency of the clearance appeared to be asymptotic as demonstrated empirically by many investigators. Actual experimental points followed the theoretical curves and verified the theory. The theory has to be extended to cope within vivo experiments.     

Immobilization of biomacromolecules onto aminolyzed poly(L-lactic acid) toward acceleration of endothelium regeneration

By reaction of poly(L-lactic acid) (PLLA) membrane with 1,6-hexanediamine, free amino groups were introduced onto a PLLA surface, through which biocompatible macromolecules such as gelatin, chitosan, or collagen were covalently immobilized by employing glutaraldehyde as a coupling agent. The existence of free amino groups on the aminolyzed PLLA surface was verified quantitatively by the ninhydrin analysis method, which revealed that surface NH(2) density increased with 1,6-hexanediamine concentration or aminolyzing time. Scanning force microscopy measurements detected an increase in surface roughness after aminolysis. The culture of human umbilical vein endothelial cells (HUVECs) in vitro proved that the cell proliferation rate and cell activity of both aminolyzed and biomacromolecule-immobilized PLLAs were improved compared with control PLLA. Scanning electron microscopy observation showed more spreading and flat cell morphology after HUVECs were cultured for 4 days on either aminolyzed or biomacromolecule-immobilized PLLA membranes. Confluent cell layers were observed on the modified PLLA. Measurement of von Willebrand factor secreted by these HUVECs confirmed that endothelium function was maintained. Therefore, aminolysis and biomacromolecule immobilization are promising ways to accelerate endothelium regeneration, which is crucial for blood vessel tissue engineering.     

Modulation of angiogenic potential of collagen matrices by covalent incorporation of heparin and loading with vascular endothelial growth factor

One of the prominent shortcomings of matrices for tissue engineering is their poor ability to support angiogenesis. We report here on experiments to enhance the angiogenic properties of collagen matrices. Our aim is to achieve this goal by covalently incorporating heparin into collagen matrices and by physically immobilizing angiogenic vascular endothelial growth factor (VEGF) to the heparin. The immobilization of heparin was performed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Carboxyl groups on the heparin are activated to succinimidyl esters, which react with amino functions on the collagen to zero length cross-links. This modification leads--in addition to the incorporation of heparin--to gross changes in in vitro degradation behavior and water-binding capacity. As a first approach to testing angiogenic capabilities, endothelial cells were exposed to nonmodified and heparinized collagen matrices. This exposure leads to an increase in endothelial cell proliferation. The increase can be further enhanced by loading the (heparinized) collagen matrices with VEGF. Evaluation of the angiogenic potential of heparinized matrices was further investigated by exposing them to the chorioallantoic membrane of chicken embryos and to the subcutaneous tissue of rats. Both approaches show that heparinized matrices have substantially increased angiogenic potential. In particular, the loading of heparinized matrices with VEGF invokes a further increase in angiogenic potential. It is apparent that the physical binding of VEGF to heparin allows for a release that is beneficial to angiogenesis. By varying the heparin and EDC/NHS concentrations during the modification process and by varying the loading with VEGF, the angiogenic potential as well as the degradation behavior can be adapted to obtain matrices that fulfill specific angiogenic requirements in the field of tissue engineering.     

Immobilization of natural macromolecules on poly-L-lactic acid membrane surface in order to improve its cytocompatibility

With the use of a grafting-coating method, three kinds of natural macromolecules, that is, gelatin, collagen, or chitosan, were immobilized on poly-L-lactic acid (PLLA) membrane surfaces with the goal of improving of cellular interactions. Attenuated total reflectance infrared spectroscopy (ATR-IR), x-ray photoelectron spectroscopy (XPS) and surface morphology analysis using scanning electronic microscopy (SEM) confirmed that the natural macromolecule layers adhered tightly to the hydrophobic PLLA membrane surfaces. Chondrocyte culture showed that the modified PLLA membranes had higher cell attachment, higher cell proliferation rate, and higher cell activity than the control PLLA membrane. Moreover, the chondrocytes were more spread out on the modified PLLA membranes than on the control PLLA membranes.     

Macromolecular release from collagen monolithic devices

Collagen monolithic devices varying in crosslinking density, collagen structure, and crosslinker were fabricated. In vitro release rates of a model macromolecule, inulin, were found to be linear with t1/2 and were affected by crosslinking density, nature of crosslinker, and collagen structure. The biodegradation of the collagen matrix was also examined. Proteolytic enzymes did not degrade the collagen devices; the degradation rate with collagenase was dependent on collagen structure, crosslinker, crosslinking density, and enzyme concentration. In vivo biocompatibility, degradation, and 14C-inulin release rates were evaluated subcutaneously in rats. After 3 weeks, none of the collagen discs induced any severe cellular response. Dacron induced a stronger fibroblast response but fewer inflammatory cells as compared to the collagen discs. No significant degradation of the collagen discs occurred within 3 weeks. In vivo release of 14C-inulin from collagen monolithic devices was diffusion controlled.