Fengfu Li

PEG-stabilized carbodiimide crosslinked collagen–chitosan hydrogels for corneal tissue engineering

Implantable biomaterials that mimic the extracellular matrix (ECM) in key physical and physiological functions require components and microarchitectures that are carefully designed to maintain the correct balance between biofunctional and physical properties. Our goal was to develop hybrid polymer networks (HPN) that combine the bioactive features of natural materials and physical characteristics of synthetic ones to achieve synergy between the desirable mechanical properties of some components with the biological compatibility and physiological relevance of others. In this study, we developed collagen-chitosan composite hydrogels as corneal implants stabilized by either a simple carbodiimide cross-linker or a hybrid cross-linking system comprised of a long-range bi-functional cross-linker (e.g. poly(ethylene glycol) dibutyraldehyde (PEG-DBA)), and short-range amide-type cross-linkers (e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS)). Optimum hybrid hydrogel demonstrated significantly enhanced mechanical strength and elasticity by 100 and 20%, respectively, compared to its non-hybrid counterpart. It demonstrated excellent optical properties, optimum mechanical properties and suturability, and good permeability to glucose and albumin. It had excellent biocompatibility and when implanted into pig corneas for 12 months, allowed seamless host-graft integration with successful regeneration of host corneal epithelium, stroma, and nerves.

Collagen-phosphorylcholine interpenetrating network hydrogels as corneal substitutes

A biointeractive collagen-phospholipid corneal substitute was fabricated from interpenetrating polymeric networks comprising 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide crosslinked porcine atelocollagen, and poly(ethylene glycol) diacrylate crosslinked 2-methacryloyloxyethyl phosphorylcholine (MPC). The resulting hydrogels showed an overall increase in mechanical strength beyond that of either original component and enhanced stability against enzymatic digestion (by collagenase) or UV degradation. More strikingly, these hydrogels retained the full biointeractive, cell friendly properties of collagen in promoting corneal cell and nerve in-growth and regeneration (despite MPCs known anti-adhesive properties). Measurements of refractive indices, white light transmission and backscatter showed the optical properties of collagen-MPC are comparable or superior to those of the human cornea. In addition, the glucose and albumin permeability were comparable to those of human corneas. Twelve-month post-implantation results of collagen-MPC hydrogels into mini-pigs showed regeneration of corneal tissue (epithelium, stroma) as well as the tear film and sensory nerves. We also show that porcine collagen can be substituted with recombinant human collagen, resulting in a fully-synthetic implant that is free from the potential risks of disease transmission (e.g. prions) present in animal source materials.

Artificial human corneas: Scaffolds for transplantation and host regeneration

Purpose To review the development of artificial corneas (pros-theses and tissue equivalents) for transplantation, and to provide recent updates on our tissue-engineered replacement corneas. Methods Modified natural polymers and synthetic polymers were screened for their potential to replace damaged portions of the human cornea or the entire corneal thickness. These polymers, combined with cells derived from each of the three main corneal layers or stem cells, were used to develop artificial corneas. Functional testing was performed in vitro. Trials of biocompatibility and immune and inflammatory reactions were performed by implanting the most promising polymers into rabbit corneas. Results Collagen-based biopolymers, combined with synthetic crosslinkers or copolymers, formed effective scaffolds for developing prototype artificial corneas that could be used as tissue replacements in the future. We have previously developed an artificial cornea that mimicked key morphologic and functional properties of the human cornea. The addition of synthetic polymers increased its toughness as it retained transparency and low light scattering, making the matrix scaffold more suitable for transplantation. These new composites were implanted into rabbits without causing any acute inflammation or immune response. We have also fabricated full-thickness composites that can be fully sutured. However, the long-term effects of these artificial corneas need to be evaluated. Conclusions Novel tissue-engineered corneas that comprise composites of natural and synthetic biopolymers together with corneal cell lines or stem cells will, in the future, replace portions of the cornea that are damaged. Our results provide a basis for the development of both implantable temporary and permanent corneal replacements.

Bioengineered corneas: how close are we?

Bioengineered corneas are substitutes for human donor tissue that are designed to replace part or the full thickness of damaged or diseased corneas. They range from prosthetic devices that solely address replacement of the corneas function to tissue-engineered hydrogels that allow some regeneration of the host tissue. In addition, there are also bioengineered lenticules that may be implanted into the cornea to improve vision by altering the refractive properties of the eye, an alternative procedure to refractive surgery. In recent years, there have been significant developments in many areas of bioengineered corneas, such as the clinical trials of an artificial cornea designed as a prosthesis, the development of completely natural corneal replacements, and the development of biosynthetic matrices that permit host tissue regeneration. For correction of refractive errors, a synthetic corneal onlay that allows stable overgrowth of epithelium appears to be promising.

Tissue-Engineered Recombinant Human Collagen-Based Corneal Substitutes for Implantation: Performance of Type I versus Type III Collagen

PURPOSE To compare the efficacies of recombinant human collagens types I and III as corneal substitutes for implantation. METHODS Recombinant human collagen (13.7%) type I or III was thoroughly mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. The final homogenous solution was either molded into sheets for in vitro studies or into implants with the appropriate corneal dimensions for transplantation into minipigs. Animals with implants were observed for up to 12 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, and fundus examination. Histopathologic examinations were also performed on corneas harvested after 12 months. RESULTS Both cross-linked recombinant collagens had refractive indices of 1.35, with optical clarity similar to that in human corneas. Their chemical and mechanical properties were similar, although RHC-III implants showed superior optical clarity. Implants into pig corneas over 12 months show comparably stable integration, with regeneration of corneal cells, tear film, and nerves. Optical clarity was also maintained in both implants, as evidenced by fundus examination. CONCLUSIONS Both RHC-I and -III implants can be safely and stably integrated into host corneas. The simple cross-linking methodology and recombinant source of materials makes them potentially safe and effective future corneal matrix substitutes.

A Collagen–Chitosan Hydrogel for Endothelial Differentiation and Angiogenesis

Cell therapy for the treatment of cardiovascular disease has been hindered by low cell engraftment, poor survival, and inadequate phenotype and function. In this study, we added chitosan to a previously developed injectable collagen matrix, with the aim of improving its properties for cell therapy and neovascularization. Different ratios of collagen and chitosan were mixed and chemically crosslinked to produce hydrogels. Swell and degradation assays showed that chitosan improved the stability of the collagen hydrogel. In culture, endothelial cells formed significantly more vascular-like structures on collagen–chitosan than collagen-only matrix. While the differentiation of circulating progenitor cells to CD31+ cells was equal on all matrices, vascular endothelial-cadherin expression was increased on the collagen–chitosan matrix, suggesting greater maturation of the endothelial cells. In addition, the collagen–chitosan matrix supported a significantly greater number of CD133+ progenitor cells than the collagen-only matrix. In vivo, subcutaneously implanted collagen–chitosan matrices stimulated greater vascular growth and recruited more von Willebrand factor (vWF+) and CXCR4+ endothelial/angiogenic cells than the collagen-only matrix. These results indicate that the addition of chitosan can improve the physical properties of collagen matrices, and enhance their ability to support endothelial cells and angiogenesis for use in cardiovascular tissue engineering applications.

Collagen and glycopolymer based hydrogel for potential corneal application.

6-Methacryloyl-alpha-D-galactopyranose (MG) was synthesized, and characterized by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectrometry, and single-crystal X-ray diffraction. A series of interpenetrating polymer network (IPN) hydrogels was fabricated by simultaneously photocuring MG crosslinked by poly(ethylene glycol) diacrylate and chemically crosslinking type I collagen with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. The successful incorporation of the glycopolymer, polymer MG, into collagen hydrogel was confirmed by FTIR and solid-state (13)C NMR. The optical characteristics of the IPN hydrogels are comparable to those of human corneas. The tensile strength and modulus of the hydrogels are enhanced by incorporation of polymer MG in comparison to that of the control collagen hydrogel. Biodegradation results indicated that polymer MG enhanced the stability of the composite hydrogels against collagenase. In vitro results demonstrated that the IPN hydrogel supported the adhesion and proliferation of human corneal epithelial cells and outperformed human cornea in blocking bacteria adhesion. Taken together, the IPN hydrogel might be a promising material for use in corneal lamellar keratoplasty.

Controlled Release of Bevacizumab Through Nanospheres for Extended Treatment of Age-Related Macular Degeneration

Bevacizumab (Avastin®) has been used by ophthalmologists in many countries as an off-label drug for the treatment of wet age-related macular degeneration (AMD). Due to its short half-life necessitating frequent intravitreal injection, a method for sustained delivery is in need. We demonstrated that bevacizumab could be released in a sustained fashion over 90 days from nano- and microspheres fabricated from poly(DL-lactide-co-glycolide) and poly(ethylene glycol)-b-poly(D,L-lactic acid), respectively. The drug release rate could be adjusted by alteration of the drug/polymer ratio. The use of such nano- and microspheres as bevacizumab delivery vehicles may improve the treatment of wet AMD.

Artificial corneas: a regenerative medicine approach

Corneal substitutes are being developed to address the shortage of human donor tissues as well as the current disadvantages in some clinical indications, which include immune rejection. In the past few years, there have been significant developments in bioengineered corneas that are designed to replace part or the full thickness of damaged or diseased corneas that range from keratoprostheses that solely address the replacement of the corneas function, through tissue-engineered hydrogels that permit regeneration of host tissues. We describe examples of corneal substitutes that encourage regeneration of the host tissue. We also contend that it is unlikely that there will be a single “one-size-fits-all” corneal substitute for all indications. Instead, there will most likely be a small range of corneal substitutes ranging from prostheses to tissue-engineered matrix substitutes that are tailored to different clusters of clinical indications. The tissue-engineered matrices can either be produced as sterile acellular matrices, or complete with functional cells, ready for implantation.

An acellular matrix-bound ligand enhances the mobilization, recruitment and therapeutic effects of circulating progenitor cells in a hindlimb ischemia model

Circulating progenitor cells home to and engraft to sites of ischemia, mediated in part by the adhesion molecule Lrselectin; however, accumulation in tissues such as the heart is low. In this study, an acellular collagen‐based matrix containing sialyl LewisX (sLeX), which binds L‐selectin, was developed in order to enhance the endogenous progenitor cell therapeutic response. Its effect on progenitor cells and angiogenesis were assessed in vitro and using a hindlimb ischemia model with rats. In culture, the sLeX– collagen matrix recruited more CD133+CD34+L‐selectm+ cells than collagen‐only matrix, with adhesion mediated by L‐selectin binding. Increased angiogenic/chemotactic cytokine production and improved resistance to apoptosis appeared in cells cultured on sLeX‐collagen matrix. In vivo, mobilization of endogenous circulating progenitor cells was increased, and greater recruitment of these and systemically injected human peripheral blood CXCR4+L‐selectin+ cells to sLeX‐collagen treated limbs was observed compared to collagen‐only. This condition was associated with differences in angiogenic/chemotactic cytokine levels, with greater arteriole density and increased perfusion in sLeX‐collagen treated hindlimbs. With these factors taken together, we demonstrated that an acellular matrix‐bound ligand approach can enhance the mobilization, recruitment, and therapeutic effects of endogenous and/or transplanted progenitor cells, possibly through paracrine and antiapoptotic mechanisms, and could be used to improve cell‐based regenerative therapies.— Suuronen, E. J., Zhang, P., Kuraitis, D., Cao, X., Melhuish, A., McKee, D., Li, F., Mesana, T. G., Veinot, J. P., Ruel, M. An acellular matrix‐bound ligand enhances the mobilization, recruitment and therapeutic effects of circulating progenitor cells in a hindlimb ischemia model. FASEBJ. 23, 1447–1458 (2009)