Initial studies using the rat sciatic nerve demonstrated the ability to adapt a multiple lumen cuff face to a nerve stump repair site. The neurons in the proximal stump grew through the individual conduits of the silicone rubber cuff, crossed a 5 mm gap, and continued into the distal stump. The effect of the cuff design on axonal regeneration was studied by comparing macroscopic and microstructural results for experimental groups of Sprague-Dawley rats with controls at 8, 12, 16, and 24 weeks post-implantation. The several individual nerve bundles which formed within the cuff lumens during these periods maintained their alignment on the distal side of the gap. The use of the multiple lumen system provided suitable scaffolding support and control of orientation and direction for fibers and established a sized, controlled environment for regeneration within each of the separate nerve cuff compartments.

Previous studies in our laboratory showed that acellular collagen scaffold implants induce tissue ingrowth and perform similar to autografts following reconstruction of rabbit Achilles tendon or anterior cruciate ligament (ACL). We chronologically review these and related studies, and report preliminary development of fibroblast-seeded collagen scaffolds potentially useful for ACL reconstruction. The ‘healing potential’ of fibroblasts was measured within collagen scaffolds in vitro, as a function of fibroblast source. Aligned collagen scaffolds were seeded with fibroblasts from rabbit ACL, synovium, patellar tendon, or skin. Fibroblast viability, adherence, spreading, proliferation, and protein and collagen deposition were measured on collagen scaffolds. The fibroblasts attached to the scaffolds, and spread along the long axis of the collagen fibers. ACL fibroblasts adhered better than other fibroblast types; however, the ACL fibroblasts proliferated at the slowest rate. Patellar tendon fibroblasts proliferated at the most rapid rate. All four of the fibroblast types secreted protein and collagen within the collagen scaffolds.

Preliminary in vivo studies suggest that fibroblasts seeded onto collagen scaffolds can remain viable following reimplantation into the donor rabbit. Ongoing studies will elucidate the role of autogenous seeded fibroblasts in neoligament formation/remodeling. These ‘ligament analogs’ are potentially useful for clinical ACL reconstruction: fibroblasts would be obtained from biopsy, cultured, seeded onto a collagen scaffold, and implanted as an ACL substitute into the same patient.

Following injuries resulting in a gap, tendons generally do not heal sufficiently because the defect does not fill with reparative tissue, or the resulting scar tissue is not functionally adequate. The objective of this study was to engineer a collagen-GAG (CG) copolymer to facilitate tendon regeneration. Previous work in our laboratory using similar porous resorbable analogs of extracellular matrix have led to regeneration of skin and peripheral nerve.

Matrices with controlled pore structure were produced by adapting the manufacturing technique developed in our laboratory for grafts with cylindrical geometry. CG suspensions contained within silicone elastomer tubes were frozen and lyophilized in a controlled manner. Quantitative optical microscopy was used to determine the percent porosity, average pore size, and pore orientation of the matrices.

The CG matrices, formed in silicone elastomer tubes, 3.8 mm in diameter, had average pore sizes ranging from 20 to 150 μm. his range of pore sizes was comparable to that obtained previously using a diameter of 1.5 mm.1 The average pore size did not appear to be strongly dependent on the tubing diameter. The homogeneity of the pore structure and the pore channel orientation could be controlled by adjusting the temperature of the bath used for freezing the CG suspension and the velocity with which the graft was immersed into the coolant bath.

Fibrous type I collagen possesses unique physicochemical, mechanical and biological properties which are useful for biomedical applications. Our research in applying collagen materials for various tissue repair has led to the development of a resorbable collagen based implant for meniscal tissue regeneration applications. We discuss here the design and development of this resorbable collagen based implant. The results of safety and efficacy studies indicate that the collagen based implant, if designed properly, can be a safe and effective template to support meniscal tissue regeneration in the knee joint.

We present a novel method for manufacturing three-dimensional, biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for use in bone regeneration. The technique involves the formation of a composite material consisting of gelatin microspheres surrounded by a PLGA matrix. The gelatin microspheres are leached out leaving an open-cell foam with a pore size and morphology defined by the gelatin microspheres. The foam porosity can be controlled by altering the volume fraction of gelatin used to make the composite material. PLGA 50:50 was used as a model degradable polymer to establish the effect of porosity, pore size, and degradation on foam mechanical properties. The compressive strengths and moduli of PLGA 50:50 foams were found to decrease with increasing porosity but were largely unaffected by pore size. Foams with compressive strengths up to 2.5 MPa were manufactured. From in vitro degradation studies we established that for PLGA 50:50 foams the mechanical properties declined in parallel with the decrease in molecular weight. Below a weight average molecular weight of 10,000 the foam had very little mechanical strength (0.02 MPa). These results indicate that PLGA 50:50 would not be suitable as a scaffold material for bone regeneration. However, the dependence of mechanical properties on porosity, pore size, and degree of degradation which we have determined will aid us in designing a PLGA foam (with a comonomer ratio other than 50:50) suitable for bone regeneration.

A processing technique based on the principle of phase separation was developed to fabricate three-dimensional microcellular foams to act as templates for cell transplantation. The polymers used to make the foams were polylactic acid (PLLA) and a polyphosphoester (BPA/PP). The resulting foams had relatively uniform, open cells throughout the matrix. The foams could also be fabricated into complex shapes to meet specific design requirements. The foam morphology and microstructure were characterized by mercury porosimetry and scanning electron microscopy. Osteoblast like cells ROS17/2.8 were successfully cultured in the foams. Cell attachment to the foam interior was verified by confocal microscopy. The fabrication technique allows incorporation of drugs or nutrients into the highly porous structure as demonstrated by the intimate dispersion of fluorescein isothiocyanate (FITC) in the matrix.

Non-woven meshes of polyglycoiic acid (PGA) fibers are attractive candidates to transplant cells and engineer new tissue. However, these devices typically lack the structural stability to resist compressive forces, and collapse in vivo. To determine whether these devices could be stabilized by physically bonding adjacent PGA fibers, we have sprayed solutions of poly (L-lactic acid) (PLLA) and a 50/50 copolymer of poly (D,L-lactic-co-glycolic acid) (PLGA) dissolved in chloroform over PGA meshes formed into hollow tubes. After the chloroform evaporated, the PLLA and PLGA formed a coating over the PGA fibers and bonded adjacent fibers. The pattern and extent of fiber bonding was found to depend on both the concentration of polymer in the spraying solution, and the total mass of polymer sprayed on the device. The compression resistance of sprayed devices increased with increasing extent of bonding, and devices bonded with PLLA resisted compressive forces better than devices bonded with PLGA. For example, under a compressive force of 200 mN, devices sprayed with PLLA compressed approximately 20%, while devices sprayed with PLGA were completely crushed. To determine whether stabilized devices such as these can guide the development of neointestine and neocartilage by transplanted cells, intestinal cells and chondrocytes have been attached to PLLA bonded devices and transplanted into experimental animals.

Collagen microspheres are used to immobilize mammalian and hybridoma cells in commercial cell culture in a continuous fluidized bed bioreactor. One advantage of this technology is the high cell densities and the resulting high reactor volume productivity. Cell densities have exceeded 108 cells per milliliter of reactor volume in steady state culture of CHO cells. The microsphere is manufactured in a multi-stage batch process that initially involves pretreating insoluble, Type I bovine hide collagen to remove fats, triglycerides and any soluble compounds. The collagen fibers are then milled and dispersed in an acid solution. Water to collagen ratios of 500/1, mass basis, have been achieved. Stainless steel weighting material is blended in the collagen dispersion in order to control the specific gravity. The dispersion is cast into droplets which are in turn frozen, thermally soaked, lyophilized, fractionated by terminal velocity, crosslinked and finally sterilized. The morphology and pore size are created during the soaking phase, wherein the completely frozen collagen dispersion is partially melted to provide a mobile phase for the collagen fibrils to rearrange. In their dispersed form, collagen fibrils have additional uses in separation and purification due to the ability to precisely control the swelling and collapsing by pH, surface area and concentration.

In the future, the development of methods for transplantation of large hepatocyte numbers could provide an alternative to the established liver and split liver transplantation. The use of three dimensional prevascularized polyvinyl alcohol (PVA) scaffolds have allowed hepatocyte transplantation equivalent to a whole liver mass. This study was designed to determine if a portacaval-shunt (PCS) or a 70% hepatectomy (70% HE) enhanced engraftment and proliferation of transplanted hepatocytes in heterotopic locations.

Male Lewis rats served as both donors and recipients, respectively. PVA-sponges were implanted as matrices for the transplanted hepatocytes. Recipient animals were divided into four groups. All groups received transplantation of 5×107 Hepatocytes (HCTx). In addition, group A received 70% HE, group B received a PCS plus 70% HE whereas group D received PCS. Group C as a control, received only HCTx. Quantitative morphometric analysis of hepatocyte area was performed on day 0, 3, 7 and 14 after transplantation. BrdU staining was performed to study DNA-Synthesis in the graft on day 3 and 14.

PCS alone and in combination with a partial hepatectomy led to significantly greater cell area one week after transplantation compared to the partial hepatectomy alone. 70% HE resulted in significantly larger cell area than the controls. At two weeks after transplantation, these significant differences persisted with the exception of Group A and C results which were not significantly different. On day 3, BrdU staining revealed a significantly higher DNA synthesis rate in Groups A and B compared to Group C. On day 14, no statistically significant differences in levels of DNA synthesis could be observed.

We conclude that hepatocytes can be successfully transplanted into PVA-devices. Engraftment and proliferation can be significantly enhanced by using portacaval shunt and partial hepatectomy as hepatotrophic stimulation.

The use of bioadhesive materials for orally-delivered, controlled release systems could increase GI transit time and improve bioavailability. Through the covalent attachment of various ligands to drug delivery systems it is possible to engineer materials to adhere to specific moieties endogenous to the alimentary mucosal layer. The entire GI tract is covered by a continuous, protective, mucous coating made of 95% water and 5% electrolytes, lipids, proteins, and glycoproteins. The GI glycoproteins consist of a protein core with covalently attached carbohydrate side chains terminating in either L-fucose or sialic acid residues. There exist several plant and animal lectins which display high affinity binding to either of these sugars.

We have studied the effect on bioadhesion of attaching biotinylated Ulex Europaeus I (UEA-I), an L-fucose specific lectin derived from the gorse seed, to barium-loaded alginate microspheres. Two carbodiimidazole (CDI) coupling procedures were investigated: premicrosphere-fabrication coupling and post-microsphere-fabrication coupling. The attachment of lectin was confirmed using an ABC reaction with HRP staining. The pre-modified microspheres clearly showed better labeling than the post-modified. For in vivo assessment of bioadhesion, 3 cohorts of 4 rats were force fed slurries of control, pre- and post-modified microspheres in saline. The animals were X-rayed 1 hour after feeding and sacrificed 19 hours later. Their entire GI tracts were excised, X-rayed and the microsphere distribution was assessed. The premodified microspheres showed greater retention in all areas of the alimentary canal. The total percentage of microspheres remaining in the GI tract after 19 hours was: 18.6% post-modified and 37.1% pre-modified. These results correlate well with the intensity of the reaction seen with the HRP staining, and suggest that attachment of mucin specific ligands to polymer microspheres may improve bioadhesion.

An intra-articular polymeric controlled release system was developed that is tailored to, and responsive to, the intensity of joint inflammation. Microspheres composed of the naturally occurring polyelectrolytes, gelatin and chondroitin sulfate, were synthesized by complex coacervation and the kinetics of release of encapsulated 14C-catalase was evaluated in vitro in the presence of inflammatory and non-inflammatory human joint fluids. The relative activity of gelatinase, a metalloprotease enzyme, was quantified in each of the joint fluids. Rate of degradation of the microspheres, and consequent release of 14C-catalase, was found to parallel the relative gelatinase activities in the joint fluids. Furthermore, various methods of crosslinking were found to affect the kinetics of microsphere degradation in the fluids. A catalase loading level of up to 28% was achieved, and the encapsulated catalase was found to retain up to 58 % of its biological activity.

The development of a computational tool to design bioerodible devices with optimal release characteristics is presented. This computational tool uses cellular automata and discrete iterations to model and simulate the release of bioactive agents (drugs) from bioerodible matrices. The simulations can accurately model surface erosion processes in multicomponent systems of arbitrary geometry and with different dissolution rates for each component. Simulation results are analyzed to show how the overall release rates are affected by the intrinsic dissolution rates, drug loading and the dispersion of the drug in the bioerodible matrix. Heterogeneities in the structure of the bioerodible matrix that can lead to local variations of erosion rates are also considered. A strongly nonlinear dependence of release rates on drug loading and dispersion is obtained and the effects of matrix heterogeneities are elucidated. Finally, guidelines are presented for screening of alternatives to minimize the development effort and experimentation required for designing devices with desired release characteristics.

Bioerodible polymers play an important role in parenteral drug delivery. They combine the advantage of controlling drug release and being absorbed by the body after fulfilling their task. The mechanism which controls drug release is the erosion of polymer bulk. In order to understand and control the release it is, therefore, essential to know the erosion mechanism of a biodegradable polymer. We found that the polyanhydrides obey a distinct erosion pattern. An erosion zone (the region where material loss occurs) moves from the surface to the center of the discs. The monomers of which these copolymers are composed also seem to play an important role in degradation and erosion.

Release of triamterene from 150–300 gm poly(DL-lactide-co-glycolide) (PLGA) microspheres was investigated in vitro as a function of lactic acid/glycolic acid (LA/GA) copolymer ratio and drug loading both with free microspheres and with microspheres embedded in a silicone matrix. Biphasic release consisting of diffusion controlled release followed by erosion controlled release corresponding to polymer degradation was observed in all samples. Drug release from PLGA 50:50 copolymer microspheres was three times faster than the release from PLGA 75:25 microspheres for the higher drug loading (20 wt%) and slightly faster for the lower drug loading (10 wt%). Release rates from spheres containing the higher drug loading were approximately one order of magnitude faster than release from spheres containing the lower drug loading for the same PLGA copolymer. The same qualitative results were observed for the spheres embedded in silicone matrices; however, the overall release was much slower. The results demonstrate that release behavior may be altered by changing LA/GA copolymer ratio, drug loading, and microsphere environment to obtain the desired release characteristics.

Present microsphere delivery system technology consisting of a single drug dispersed within a polymer matrix has several drawbacks. One is the problem of the so-called “burst effect”. By exploiting the phenomenon of phase separation between two immiscible polymers dissolved in a mutual solvent, a double-walled microsphere could be manufactured with the second polymer coating the polymer/drug matrix. This one-step process would give a consistent coating of even very small microspheres not achievable via normal, two-step coating processes and would help to smooth out the release curve by lessening the “burst effect”. Along with solving the problem of the “burst effect”, this concept of double-walled microspheres could be used to achieve constant release of the drug over long periods of time. So far, this has only been achieved with a limited number of geometric configurations. Since every polymer has its own characteristic release rate, the release could be kept much more constant by changing the polymer type and/or properties. By combining these layers so that the release rate of one layer would complement the slowing of release due to decreased surface area or increased diffusion distances.

In an attempt to improve endothelial cell adhesion and growth on a polyurethane copolymer, cell adhesive RGD-containing peptides were grafted to the polymer backbone. Two peptide grafting reaction schemes, including one-step and two-step approaches, were developed. Amino acid analysis confirmed that the two-step approach had a higher peptide coupling efficiency. The two-step reaction scheme was utilized to prepare GRGDSY, GRGDVY and GRGESY (inactive control) peptide grafted polyurethanes with two different peptide densities (100 and 250 μmol/g polymer). Dynamic contact angle measurements indicated that the surfaces of the peptide grafted polyurethanes were more hydrophilic than the starting and carboxylated versions of the precursor polyurethane. In-vitro endothelial cell adhesion experiments showed that, without the presence of serum in culture medium, the GRGDSY- and GRGDVY-grafted polyurethanes dramatically enhanced cell attachment and spreading. Increasing the peptide density from 100 to 250 μmol/g polymer for the GRGDSYand GRGDVY-grafted polyurethanes resulted in an increase in cell attachment. With approximately the same peptide density (100 or 250 μmol/g polymer), the GRGDVY-grafted polymers supported more adherent cells than the GRGDSY-grafted polymers. Similar trends were observed in the in-vitro endothelial cell growth studies using culture medium containing serum and endothelial cell growth supplement. These RGD-peptide grafted polyurethanes may be useful in providing an easily prepared cell-adhesive substrate for various implantable devices and hybrid organs.

Adhesion, proliferation and motility of bovine pulmonary artery endothelial cells and of rat calvarial osteoblasts were examined in vitro and on glass surfaces modified with immobilized bioactive peptides. The peptides Arginine-Glycine-Aspartic Acid-Serine (RGDS), Arginine-Aspartic Acid-Glycine-Serine (RDGS), and Tyrosine-Isoleucine-Glycine-Serine-Arginine-Glycine (YIGSRG) were covalently bound to aminophase glass. The results of this study showed that modification of the substrate surface with immobilized peptides affected each cell line in different ways. Incorporation of this knowledge in the design of implant materials could result in biomaterials which promote and/or sustain a number of desirable cellular functions at the tissue-implant interface.

We have investigated the adhesion of rat osteoblasts on biodegradable poly(α-hydroxy ester) films as an in vitro model of bone regeneration. Osteoblasts cultured on poly(L-lactic acid), poly(DL-lactic-co-glycolic acid) copolymers, and poly(glycolic acid) films for 14 days grew with rates comparable to those observed for tissue culture polystyrene and retained their phenotype as expressed by activity of alkaline phosphatase and collagen synthesis.

An understanding of bone cell response and extracellular matrix production to a biomaterial is crucial for development of new prosthetic devices. The nature of the cellular-biomaterial surface interface will depend upon a number of factors including substrate properties (surface chemistry, charge, topography) as well as biological cellular concerns (i.e. adsorption of attachment factors to the surface, growth factors). The quality of the matrix and bone-bonding may be influenced by these factors. Recently, a short-term in-vitro cell culture assay has demonstrated the initial attachment and spread of human derived bone cells on metallic (titanium and stainless steel) and polymeric surfaces to be dependent on the adsorption of adhesive attachment factor proteins (fibronectin and vitronectin) to the substratum surface [1]. The morphological appearance of human osteoblasts cultured on titanium and stainless steel with time also demonstrated differences compared to tissue culture plastic [2]. Little data however, is available for the mitogenic and gene expression levels of primary human bone cells cultured on commonly used orthopaedic materials and the response of these cells to growth factors. The present study examined the mitogenic response and steady state mRNA expression levels of primary human bone cells cultured on metallic substrates to provide further insight into the nature of cell-substrate interactions.

Cellular interactions at the interface between bone and a prosthetic device plays an important role in total joint reconstruction success or failure. Aseptic loosening is a typical long-term complication encountered due to a failure at this interface. A number of factors have been shown to influence cell mitogenic and morphological behavior including load and stability [1] surface topography and surface chemistry [2]. The selection of suitable surface coatings on a prosthetic device would be facilitated if the nature of the interaction of bone cells to surface could be predicted from appropriate behavior of cells in tissue culture [3]. Molecular biology techniques now enable investigators to study cell-substrate interactions and gene expression at the mRNA level.