Osteochondral defect repair using bilayered hydrogels encapsulating both chondrogenically and osteogenically pre-differentiated mesenchymal stem cells in a rabbit model.

OBJECTIVE: To investigate the ability of cell-laden bilayered hydrogels encapsulating chondrogenically and osteogenically (OS) pre-differentiated mesenchymal stem cells (MSCs) to effect osteochondral defect repair in a rabbit model. By varying the period of chondrogenic pre-differentiation from 7 (CG7) to 14 days (CG14), the effect of chondrogenic differentiation stage on osteochondral tissue repair was also investigated. METHODS: Rabbit MSCs were subjected to either chondrogenic or osteogenic pre-differentiation, encapsulated within respective chondral/subchondral layers of a bilayered hydrogel construct, and then implanted into femoral condyle osteochondral defects. Rabbits were randomized into one of four groups (MSC/MSC, MSC/OS, CG7/OS, and CG14/OS; chondral/subchondral) and received two similar constructs bilaterally. Defects were evaluated after 12 weeks. RESULTS: All groups exhibited similar overall neo-tissue filling. The delivery of OS cells when compared to undifferentiated MSCs in the subchondral construct layer resulted in improvements in neo-cartilage thickness and regularity. However, the addition of CG cells in the chondral layer, with OS cells in the subchondral layer, did not augment tissue repair as influenced by the latter when compared to the control. Instead, CG7/OS implants resulted in more irregular neo-tissue surfaces when compared to MSC/OS implants. Notably, the delivery of CG7 cells, when compared to CG14 cells, with OS cells stimulated morphologically superior cartilage repair. However, neither osteogenic nor chondrogenic pre-differentiation affected detectable changes in subchondral tissue repair. CONCLUSIONS: Cartilage regeneration in osteochondral defects can be enhanced by MSCs that are chondrogenically and osteogenically pre-differentiated prior to implantation. Longer chondrogenic pre-differentiation periods, however, lead to diminished cartilage repair.

Biomimetic modification of synthetic hydrogels by incorporation of adhesive peptides and calcium phosphate nanoparticles: in vitro evaluation of cell behavior.

The ultimate goal of this work was to develop a biocompatible and biomimetic in situ crosslinkable hydrogel scaffold with an instructive capacity for bone regenerative treatment. To this end, synthetic hydrogels were functionalized with two key components of the extracellular matrix of native bone tissue, i.e. the three-amino acid peptide sequence RGD (which is the principal integrin-binding domain responsible for cell adhesion and survival of anchorage-dependent cells) and calcium phosphate (CaP) nanoparticles in the form of hydroxyapatite (which are similar to the inorganic phase of bone tissue). Rat bone marrow osteoblast-like cells (OBLCs) were encapsulated in four different biomaterials (plain oligo(poly(ethylene glycol) fumarate) (OPF), RGD-modified OPF, OPF enriched with CaP nanoparticles and RGD-modified OPF enriched with CaP nanoparticles) and cell survival, cell spreading, proliferation and mineralized matrix formation were determined via cell viability assay, histology and biochemical analysis for alkaline phosphatase activity and calcium. This study showed that RGD peptide sequences promoted cell spreading in OPF hydrogels and hence play a crucial role in cell survival during the early stage of culture, whereas CaP nanoparticles significantly enhanced cell-mediated hydrogel mineralization. Although cell spreading and proliferation activity were inhibited, the combined effect of RGD peptide sequences and CaP nanoparticles within OPF hydrogel systems elicited a better biological response than that of the individual components. Specifically, both a sustained cell viability and mineralized matrix production mediated by encapsulated OBLCs were observed within these novel biomimetic composite systems.

Advances in biomaterials for skeletal muscle engineering and obstacles still to overcome.

Repair of injured skeletal muscle is a sophisticated process that uses immune, muscle, perivascular, and neural cells. In acute injury, the robust endogenous repair process can facilitate complete regeneration with little to no functional deficit. However, in severe injury, the damage is beyond the capacity for self-repair, often resulting in structural and functional deficits. Aside from the insufficiencies in muscle function, the aesthetic deficits can impact quality of life. Current clinical treatments are significantly limited in their capacity to structurally and functionally repair the damaged skeletal muscle. Therefore, alternative approaches are needed. Biomaterial therapies for skeletal muscle engineering have leveraged natural materials with sophisticated scaffold fabrication techniques to guide cell infiltration, alignment, and differentiation. Advances in biomaterials paired with a standardized and rigorous assessment of resulting tissue formation have greatly advanced the field of skeletal muscle engineering in the last several years. Herein, we discuss the current trends in biomaterials-based therapies for skeletal muscle regeneration and present the obstacles still to be overcome before clinical translation is possible. With millions of people affected by muscle trauma each year, the development of a therapy that can repair the structural and functional deficits after severe muscle injury is pivotal.

Modular, tissue-specific, and biodegradable hydrogel cross-linkers for tissue engineering.

Synthetic hydrogels are investigated extensively in tissue engineering for their tunable physicochemical properties but are bioinert and lack the tissue-specific cues to produce appropriate biological responses. To introduce tissue-specific biochemical cues to these hydrogels, we have developed a modular hydrogel cross-linker, poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT), that can be functionalized with small peptide-based cues and large macromolecular cues simply by mixing PdBT in water with the appropriate biomolecules at room temperature. Cartilage- and bone-specific PdBT macromers were generated by functionalization with a cartilage-associated hydrophobic N-cadherin peptide, a hydrophilic bone morphogenetic protein peptide, and a cartilage-derived glycosaminoglycan, chondroitin sulfate. These biofunctionalized PdBT macromers can spontaneously cross-link polymers such as poly(N-isopropylacrylamide) to produce rapidly cross-linking, highly swollen, cytocompatible, and hydrolytically degradable hydrogels suitable for mesenchymal stem cell encapsulation. These favorable properties, combined with PdBT's modular design and ease of functionalization, establish strong potential for its usage in tissue engineering applications.

Effect of macromer molecular weight on in vitro ophthalmic drug release from photo-crosslinked matrices.

The objective of this study was to investigate the effect of poly(propylene fumarate) (PPF) molecular weight on the release kinetics of two ophthalmic model drugs, acetazolamide (AZ) and timolol maleate (TM), from matrices prepared by photo-induced copolymerization of PPF with N-vinyl pyrrolidone (NVP). PPF macromers of different number average molecular weight (M(n)) and polydispersity index (PI) were used in the experiments. Photo-crosslinked matrices were loaded with 5wt.% AZ or TM. The amounts of released drug and NVP were determined using high-performance liquid chromatography (HPLC). The release kinetics of both drugs was influenced by the molecular weight of the constituent PPF macromer. An increased M(n) led to an increased burst release and an accelerated drug release. Dependent on the PPF M(n), the initial AZ loading was released within periods ranging from 35 days (M(n) = 3670, PI = 1.9) to 220 days (M(n) = 2050, PI=1.5). TM-loaded matrices revealed similar release kinetics dependent on the PPF M(n). The amount of released NVP from photo-crosslinked matrices during the course of a release experiment was independent of the PPF M(n) for both drugs. Matrix swelling and erosion were determined gravimetrically. The network structures of non-loaded matrices were further characterized by determining their crosslinking densities and the relative double bond conversions of fumaric acid (FAA) and NVP. Independent of PPF M(n), PPF and NVP similarly participated in the formation of the PPF/polyNVP copolymer network. The observed differences in drug release might therefore be explained by differences in the microstructural organization of the copolymer networks. Overall, the results demonstrate that drug release kinetics from photo-crosslinked PPF/polyNVP matrices is strongly dependent on the M(n) of the PPF macromer.

Degradation and biocompatibility of a poly(propylene fumarate)-based/alumoxane nanocomposite for bone tissue engineering.

In this work, we evaluated the in vitro cytotoxicity and in vivo biocompatibility of a novel poly(propylene fumarate) (PPF)-based/alumoxane nanocomposite for bone tissue engineering applications. The incorporation of functionalized alumoxane nanoparticles into the PPF-based polymer was previously shown to significantly increase the material's flexural mechanical properties. In the current study, samples underwent accelerated in vitro degradation to allow the study of biological responses to these materials over the course of their degradation on a shortened timescale. The polymer, a macrocomposite composed of the polymer and micron-sized particles, and the nanocomposite were evaluated at three stages of degradation for in vitro cytotoxicity with a fibroblast cell line and in vivo soft-tissue response after 3 and 12 weeks of implantation in adult goats. All three material groups experienced mass loss during degradation, but the nanocomposite group eroded significantly faster than the other groups. Nondegraded materials demonstrated minimal cytotoxicity and a minor inflammatory response in soft tissue. On the contrary, predegraded samples elicited more pronounced responses, though these were due to the increase in surface area, surface roughness, and fragmentation associated with the degradation process. The presence of alumoxane nanoparticles in the PPF-based nanocomposite did not significantly affect its cytotoxicity or biocompatibility.

Injectable, in situ forming poly(propylene fumarate)-based ocular drug delivery systems.

This study sought to develop an injectable formulation for long-term ocular delivery of fluocinolone acetonide (FA) by dissolving the anti-inflammatory drug and the biodegradable polymer poly(propylene fumarate) (PPF) in the biocompatible, water-miscible, organic solvent N-methyl-2-pyrrolidone (NMP). Upon injection of the solution into an aqueous environment, a FA-loaded PPF matrix is precipitated in situ through the diffusion/extraction of NMP into surrounding aqueous fluids. Fabrication of the matrices and in vitro release studies were performed in phosphate buffered saline at 37 degrees C. Drug loadings up to 5% were achieved. High performance liquid chromatography was employed to determine the released amount of FA. The effects of drug loading, PPF content of the injectable formulation, and additional photo-crosslinking of the matrix surface were investigated. Overall, FA release was sustained in vitro over up to 400 days. After an initial burst release of 22 to 68% of initial FA loading, controlled drug release driven by diffusion and bulk erosion was observed. Drug release rates in a therapeutic range were demonstrated. Release kinetics were found to be dependent on drug loading, formulation PPF content, and extent of surface crosslinking. The results suggest that injectable, in situ formed PPF matrices are promising candidates for the formulation of long-term, controlled delivery devices for intraocular drug delivery.

Long-term release of fluocinolone acetonide using biodegradable fumarate-based polymers.

Intraocular drug delivery systems made from biodegradable polymers hold great potential to effectively treat chronic diseases of the posterior segment of the eye. This study is based on the hypothesis that crosslinked poly(propylene fumarate) (PPF)-based matrices are suitable long-term delivery devices for the sustained release of the anti-inflammatory drug fluocinolone acetonide (FA) due to their hydrophobicity and network density. FA-loaded rods of 10 mm length and 0.6 mm diameter were fabricated by photo-crosslinking PPF with N-vinyl pyrrolidone (NVP). The released amounts of FA and NVP were determined by HPLC analysis. The effects of drug loading and the ratio of PPF to NVP on the release kinetics were investigated using a 2(3-1) factorial design. Overall, FA release was sustained in vitro over almost 400 days by all tested formulations. Low burst release was followed by a dual modality release controlled by diffusion and bulk erosion with release rates up to 1.7 microg/day. The extent of the burst effect and the release kinetics were controlled by the drug loading and the matrix composition. Matrix water content and degradation were determined gravimetrically. Micro-computed tomography was used to image structural and dimensional changes of the devices. The results show that photo-crosslinked PPF-based matrices are promising long-term delivery devices for intraocular drug delivery.

A rapid, flexible method for incorporating controlled antibiotic release into porous polymethylmethacrylate space maintainers for craniofacial reconstruction.

Severe injuries in the craniofacial complex, resulting from trauma or pathology, present several challenges to functional and aesthetic reconstruction. The anatomy and position of the craniofacial region make it vulnerable to injury and subsequent local infection due to external bacteria as well as those from neighbouring structures like the sinuses, nasal passages, and mouth. Porous polymethylmethacrylate (PMMA) "space maintainers" have proven useful in staged craniofacial reconstruction by promoting healing of overlying soft tissue prior to reconstruction of craniofacial bones. We describe herein a method by which the porosity of a prefabricated porous PMMA space maintainer, generated by porogen leaching, can be loaded with a thermogelling copolymer-based drug delivery system. Porogen leaching, space maintainer prewetting, and thermogel loading all significantly affected the loading of a model antibiotic, colistin. Weeks-long release of antibiotic at clinically relevant levels was achieved with several formulations. In vitro assays confirmed that the released colistin maintained its antibiotic activity against several bacterial targets. Our results suggest that this method is a valuable tool in the development of novel therapeutic approaches for the treatment of severe complex, infected craniofacial injuries.

Injectable dual-gelling cell-laden composite hydrogels for bone tissue engineering.

The present work investigated the osteogenic potential of injectable, dual thermally and chemically gelable composite hydrogels for mesenchymal stem cell (MSC) delivery in vitro and in vivo. Composite hydrogels comprising copolymer macromers of N-isopropylacrylamide were fabricated through the incorporation of gelatin microparticles (GMPs) as enzymatically digestible porogens and sites for cellular attachment. High and low polymer content hydrogels with and without GMP loading were shown to successfully encapsulate viable MSCs and maintain their survival over 28 days in vitro. GMP incorporation was also shown to modulate alkaline phosphatase production, but enhanced hydrogel mineralization along with higher polymer content even in the absence of cells. Moreover, the regenerative capacity of 2 mm thick hydrogels with GMPs only, MSCs only, or GMPs and MSCs was evaluated in vivo in an 8 mm rat critical size cranial defect for 4 and 12 weeks. GMP incorporation led to enhanced bony bridging and mineralization within the defect at each timepoint, and direct bone-implant contact as determined by microcomputed tomography and histological scoring, respectively. Encapsulation of both GMPs and MSCs enabled hydrogel degradation leading to significant tissue infiltration and osteoid formation. The results suggest that these injectable, dual-gelling cell-laden composite hydrogels can facilitate bone ingrowth and integration, warranting further investigation for bone tissue engineering.

Controlled release of rhBMP-2 loaded poly(dl-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo.

The release kinetics of recombinant human bone morphogenetic protein-2 (rhBMP-2) loaded poly(dl-lactic-co-glycolic acid)/calcium phosphate cement (PLGA/Ca-P cement) composites were studied in vivo. RhBMP-2 was radiolabeled with (131)I and entrapped within PLGA microparticles or adsorbed onto the microparticle surface. PLGA microparticles were prepared of high molecular weight (HMW) PLGA (weight average molecular weight [M(w)] 49,100+/-1700) or low molecular weight (LMW) PLGA (M(w) 5,900+/-300) and used for preparation of 30:70 wt.% PLGA/Ca-P cement composite discs. Release of 131I-rhBMP-2 loaded composites was assessed by scintigraphic imaging according to a 2(2) two-level full factorial design in the rat ectopic model during four weeks. In vivo release kinetics varied among formulations. All formulations showed slow release without initial burst, and displayed a linear release from 3 to 28 days. Release of LMW entrapped rhBMP-2 composites (1.7+/-0.3%/day) was significantly faster than release from other formulations (p<0.01). After 28 days, retention within the composites was 65+/-5%, 75+/-4%, 50+/-4% and 70+/-6% of the initial rhBMP-2 for HMW entrapped, HMW adsorbed, LMW entrapped and LMW adsorbed rhBMP-2 composites, respectively. Release from the composite was probably slowed down by an interaction of rhBMP-2 and Ca-P cement after rhBMP-2 release from PLGA microparticles. We conclude that PLGA/Ca-P cement composites can be considered as sustained slow release vehicles and that the release and retention of rhBMP-2 can be modified according to the desired profile to a limited extent.

Growth factor-loaded scaffolds for bone engineering.

The objective of the study presented here was to investigate the bone inductive properties as well as release kinetics of rhTGF-beta1- and rhBMP-2-loaded Ti-fiber mesh and CaP cement scaffolds. Therefore, Ti-fiber mesh and porous CaP cement scaffolds were provided with these growth factors and inserted in subcutaneous and cranial implant locations in rats and rabbits. In vitro, a rapid release of rhTGF-beta1 was observed during the first 2 h of the Ti-fiber mesh scaffolds. During this time, more than 50% of the total dose of rhTGF-beta1 was released. Following this initial peak, a decline in the level of rhTGF-beta1 occurred. After 1 week, the entire theoretical initial dose was observed to have been released. This in contrast to the rhTGF-beta1 and rhBMP-2 release of the porous CaP cement scaffolds. Here, no substantial initial burst release was observed. The scaffolds showed an initial release of about 1% after 1 day, followed by an additional marginal release after 1 week. Histological analysis revealed excellent osteoconductive properties of non-loaded Ca-P material. Inside non-loaded Ti-mesh fiber scaffolds, also bone ingrowth occurred. Quantification of the bone ingrowth showed that bone formation was increased significantly in all scaffold materials by administration of rhTGF-beta1 and rhBMP-2. Consequently, we conclude that the release kinetics of growth factors from porous CaP cement differs from other scaffold materials, like metals and polymers. Nevertheless, orthotopic bone formation in a rabbit cranial defect model was stimulated in rhTGF-beta1- and rhBMP-2-loaded CaP cement and Ti-fiber mesh scaffolds compared with non-loaded implants.

Flory interaction parameter chi for hydrophilic copolymers with water.

In biomedical applications of hydrophilic polymers knowledge is required of the thermodynamic interactions between the candidate biomaterial and biological fluids. Since information on these interactions is not readily available, a new method is proposed here to estimate the copolymer-water Flory interaction parameter, chi, for biomaterials. The method is based on the pairwise thermodynamic interactions of copolymer segments and solvent molecules. It is validated using data for an important biomedical hydrogel, poly(2-hydroxyethyl methacrylate-co-methacrylic acid), in contact with water.

Prediction of feed comonomer and solvent composition for monomer-free polymer production.

A method has been developed to establish the desirable polymerization conditions for the production of monomer-free polymer at 100% conversion. The limits of desirable comonomer composition and solvent content in the feed are determined for both bulk and solution homo- and copolymerizations. An example of the result of the polymerization of 2-hydroxyethyl methacrylate and methyl methacrylate is used to illustrate the application of this method.

Injectable biodegradable materials for orthopedic tissue engineering.

The large number of orthopedic procedures performed each year, including many performed arthroscopically, have led to great interest in injectable biodegradable materials for regeneration of bone and cartilage. A variety of materials have been developed for these applications, including ceramics, naturally derived substances and synthetic polymers. These materials demonstrate overall biocompatibility and appropriate mechanical properties, as well as promote tissue formation, thus providing an important step towards minimally invasive orthopedic procedures. This review provides a comparison of these materials based on mechanical properties, biocompatibility and regeneration efficacy. Advantages and disadvantages of each material are explained and design criteria for injectable biodegradable systems are provided.

Review: tissue engineering for regeneration of articular cartilage.

Joint pain due to cartilage degeneration is a serious problem, affecting people of all ages. Although many techniques, often surgical, are currently employed to treat this affliction, none have had complete success. Recent advances in biology and materials science have pushed tissue engineering to the forefront of new cartilage repair techniques. This review seeks to condense information for the biomaterialist interested in developing materials for this application. Articular cartilage anatomy, types of injury, and current repair methods are explained. The need for biomaterials, current commonly used materials for tissue-engineered cartilage, and considerations in scale-up of cell-biomaterial constructs are summarized.

Retinal pigment epithelium engineering using synthetic biodegradable polymers.

Retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina, especially photoreceptors. Alteration in RPE structure and function is implicated in a variety of ocular disorders. Tissue engineering strategies using synthetic biodegradable polymers as temporary substrates for RPE cell culture and subsequent transplantation may provide a promising new therapy. In this review article, the manufacture of thin biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) films and their degradation behavior in vitro are discussed. RPE cell proliferation and differentiation on these PLGA films are reviewed. The fabrication of model substrates with desired chemical micropatterns in the micrometer scale is discussed and the effects of surface patterning on RPE morphology and function are assessed. Finally. the preparation of biodegradable micropatterns with adhesive PLGA and non-adhesive poly(ethylene glycol)/PLA domains to modulate RPE cell adhesion is presented.

Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability.

Poly(ethylenimine) (PEI) was used to transfect the endothelial cell line EA.hy 926, and the secreted levels of three gene products, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), and von Willebrand Factor (vWF), were assessed via ELISA. We found that the levels of these gene products in cell supernatants increased by factors up to 16.3 (tPA), 8.3 (PAI-1), or 6.7 (vWF) times the levels recorded for untreated cells, and roughly correlated with the percentage of cells that expressed the reporter plasmid. Transfections carried out using promotorless constructs of the same reporter plasmid also yielded increases in tPA, PAI-1, and vWF to similar extents. Additionally, data regarding cell viability were gathered and found to inversely relate to both the effectiveness of the PEI used for transfection and the secreted levels of the three mentioned products. There appeared to be two distinct types of cell death, resulting from the use of either free PEI (which acts within 2 h) or PEI/DNA complexes (which cause death 7-9 h after transfection). Cells were also transfected by poly(L-lysine) and liposomal carriers, and increases in secreted tPA similar to those seen with PEI-mediated transfection were observed for positively transfected cells. The results of these investigations indicate that non-viral gene delivery can induce a state of endothelial cell dysfunction, and that PEI-mediated transfection can lead to two distinct types of cell death.