Reduction of the inflammatory responses against alginate-poly-L-lysine microcapsules by anti-biofouling surfaces of PEG-b-PLL diblock copolymers.

Large-scale application of alginate-poly-L-lysine (alginate-PLL) capsules used for microencapsulation of living cells is hampered by varying degrees of success, caused by tissue responses against the capsules in the host. A major cause is proinflammatory PLL which is applied at the surface to provide semipermeable properties and immunoprotection. In this study, we investigated whether application of poly(ethylene glycol)-block-poly(L-lysine hydrochloride) diblock copolymers (PEG-b-PLL) can reduce the responses against PLL on alginate-matrices. The application of PEG-b-PLL was studied in two manners: (i) as a substitute for PLL or (ii) as an anti-biofouling layer on top of a proinflammatory, but immunoprotective, semipermeable alginate-PLL100 membrane. Transmission FTIR was applied to monitor the binding of PEG-b-PLL. When applied as a substitute for PLL, strong host responses in mice were observed. These responses were caused by insufficient binding of the PLL block of the diblock copolymers confirmed by FTIR. When PEG-b-PLL was applied as an anti-biofouling layer on top of PLL100 the responses in mice were severely reduced. Building an effective anti-biofouling layer required 50 hours as confirmed by FTIR, immunocytochemistry and XPS. Our study provides new insight in the binding requirements of polyamino acids necessary to provide an immunoprotective membrane. Furthermore, we present a relatively simple method to mask proinflammatory components on the surface of microcapsules to reduce host responses. Finally, but most importantly, our study illustrates the importance of combining physicochemical and biological methods to understand the complex interactions at the capsules' surface that determine the success or failure of microcapsules applicable for cell-encapsulation.

Synthesis and Phase Behavior of Poly(N-isopropylacrylamide)-b- Poly(L-Lysine Hydrochloride) and Poly(N-Isopropylacrylamide- co-Acrylamide)-b-Poly(L-Lysine Hydrochloride).

The synthesis of poly(N-isopropylacrylamide)-b-poly(L-lysine) and poly(N- isopropylacrylamide-co-acrylamide)-b-poly(L-lysine) copolymers was accomplished by combining atom transfer radical polymerization (ATRP) and ring opening polymerization (ROP). For this purpose, a di-functional initiator with protected amino group was successfully synthetized. The ATRP of N-isopropylacrylamide yielded narrowly dispersed polymers with consistent high yields (~80%). Lower yields (~50%) were observed when narrowly dispersed random copolymers of N-isopropylacrylamide and acrylamide where synthesized. Amino-terminated poly(N-isopropylacrylamide) and poly(N-isopropylacrylamide- co-acrylamide) were successfully used as macroinitiators for ROP of N6-carbobenzoxy-L- lysine N-carboxyanhydride. The thermal behavior of the homopolymers and copolymers in aqueous solutions was studied by turbidimetry, dynamic light scattering (DLS) and proton nuclear magnetic resonance spectroscopy (¹H-NMR).

Changes in articular cartilage after meniscectomy and meniscus replacement using a biodegradable porous polymer implant.

PURPOSE: To evaluate the long-term effects of implantation of a biodegradable polymer meniscus implant on articular cartilage degeneration and compare this to articular cartilage degeneration after meniscectomy. METHODS: Porous polymer polycaprolacton-based polyurethane meniscus implants were implanted for 6 or 24 months in the lateral compartment of Beagle dog knees. Contralateral knees were meniscectomized, or left intact and served as controls. Articular cartilage degeneration was evaluated in detail using India ink staining, routine histology, immunochemistry for denatured (Col2-¾M) and cleaved (Col2-¾C(short)) type II collagen, Mankin's grading system, and cartilage thickness measurements. RESULTS: Histologically, fibrillation and substantial immunohistochemical staining for both denatured and cleaved type II collagen were found in all three treatment groups. The cartilage of the three groups showed identical degradation patterns. In the 24 months implant group, degradation appeared to be more severe when compared to the 6 months implant group and meniscectomy group. Significantly more cartilage damage (India ink staining, Mankin's grading system, and cartilage thickness measurements) was found in the 24 months implant group compared to the 6 months implant group and meniscectomy group. CONCLUSION: Degradation of the cartilage matrix was the result of both mechanical overloading as well as localized cell-mediated degradation. The degeneration patterns were highly variable between animals. Clinical application of a porous polymer implant for total meniscus replacement is not supported by this study.

Polyacylurethanes as Novel Degradable Cell Carrier Materials for Tissue Engineering.

Polycaprolactone (PCL) polyester and segmented aliphatic polyester urethanes based on PCL soft segment have been thoroughly investigated as biodegradable scaffolds for tissue engineering. Although proven beneficial as long term implants, these materials degrade very slowly and are therefore not suitable in applications in which scaffold support is needed for a shorter time. A recently developed class of polyacylurethanes (PAUs) is expected to fulfill such requirements. Our aim was to assess in vitro the degradation of PAUs and evaluate their suitability as temporary scaffold materials to support soft tissue repair. With both a mass loss of 2.5-3.0% and a decrease in molar mass of approx. 35% over a period of 80 days, PAUs were shown to degrade via both bulk and surface erosion mechanisms. Fourier Transform Infra Red (FTIR) spectroscopy was successfully applied to study the extent of PAUs microphase separation during in vitro degradation. The microphase separated morphology of PAU1000 (molar mass of the oligocaprolactone soft segment = 1000 g/mol) provided this polymer with mechano-physical characteristics that would render it a suitable material for constructs and devices. PAU1000 exhibited excellent haemocompatibility in vitro. In addition, PAU1000 supported both adhesion and proliferation of vascular endothelial cells and this could be further enhanced by pre-coating of PAU1000 with fibronectin (Fn). The contact angle of PAU1000 decreased both with in vitro degradation and by incubation in biological fluids. In endothelial cell culture medium the contact angle reached 60°, which is optimal for cell adhesion. Taken together, these results support the application of PAU1000 in the field of soft tissue repair as a temporary degradable scaffold.

Effect on tissue differentiation and articular cartilage degradation of a polymer meniscus implant: A 2-year follow-up study in dogs.

BACKGROUND: Replacement of the meniscus by an implant could potentially avoid cartilage degeneration. HYPOTHESIS: An implant of degradable polycaprolacton-polyurethane should act as a temporary scaffold enabling regeneration of a new meniscus by slow degradation of the polymer and simultaneous in-growth and differentiation of tissues into the typical cartilage-like tissue of the meniscus. STUDY DESIGN: Controlled laboratory study. METHODS: In 13 dogs' knees, the lateral meniscus was replaced with a porous polymer implant (6 and 7 for 6- and 24-month follow-up, respectively); in 7 knees only a meniscectomy was performed. In 6 knees, no surgery was performed. After 6 and 24 months, the implants and the articular cartilage were histologically evaluated. Compression-stress tests were performed on implant biopsy specimens. RESULTS: The implants were fully integrated into the tissue without formation of a capsule. The foreign body reaction did not exceed grade I. Differentiation from fibrous- to cartilage-like tissue was pronounced after 24 months. Viable cells were particularly absent after 24 months in central parts of the most anterior part of the scaffold. The mechanical properties of the implants were intermediate between the scaffold before implantation and native meniscus tissue and were not different between 6 and 24 months. After both 6 and 24 months, small areas of the implant were not covered with tissue. Cartilage degeneration was not prevented. CONCLUSION: A final remodeling of tissue into neomeniscus tissue could not take place since the original structure of the polymer was still present after 24 months. The implant did not prevent cartilage degradation. Several factors are discussed that may be responsible for this. CLINICAL RELEVANCE: Although clinical application of a polymer implant for the replacement of the entire meniscus is not supported by this study, the authors strongly believe in the concept, but further improvements in the implant and surgical technique are needed before such an implant can be recommended for human clinical use.

Replacement of the knee meniscus by a porous polymer implant: a study in dogs.

BACKGROUND: Meniscectomy will lead to articular cartilage degeneration in the long term. Therefore, the authors developed an implant to replace the native meniscus. HYPOTHESIS: The porous polymer meniscus implant develops into a neomeniscus and protects the cartilage from degeneration. STUDY DESIGN: Controlled laboratory study. METHODS: In a dog model, a porous polymer scaffold with optimal properties for tissue infiltration and regeneration of a neomeniscus was implanted and compared with total meniscectomy. The tissue infiltration and redifferentiation in the scaffold, the stiffness of the scaffold, and the articular cartilage degeneration were evaluated. RESULTS: Three months after implantation, the implant was completely filled with fibrovascular tissue. After 6 months, the central areas of the implant contained cartilage-like tissue with abundant collagen type II and proteoglycans in their matrix. The foreign-body reaction remained limited to a few giant cells in the implant. The compression modulus of the implant-tissue construct still differed significantly from that of the native meniscus, even at 6 months. Cartilage degeneration was observed both in the meniscectomy group and in the implant group. CONCLUSION: The improved properties of these polymer implants resulted in a faster tissue infiltration and in phenotypical differentiation into tissue resembling that of the native meniscus. However, the material characteristics of the implant need to be improved to prevent degeneration of the articular cartilage. CLINICAL RELEVANCE: The porous polymer implant developed into a polymer-tissue construct that resembled the native meniscus, and with improved gliding characteristics, this prosthesis might be a promising implant for the replacement of the meniscus.

Assessment of tissue ingrowth rates in polyurethane scaffolds for tissue engineering.

The continuous development of new biomaterials for tissue engineering and the enhancement of tissue ingrowth into existing scaffolds, using growth factors, create the necessity for developing adequate tools to assess tissue ingrowth rates into porous biomaterials. Current histomorphometric techniques evaluating rates of tissue ingrowth tend either to measure the overall tissue content in an entire sample or to depend on the user to indicate a front of tissue ingrowth. Neither method is particularly suitable for the assessment of tissue ingrowth rates, as these methods either lack the sensitivity required or are problematic when there is a tissue ingrowth gradient rather than an obvious tissue ingrowth front. This study describes a histomorphometric method that requires little observer input, is sensitive, and renders detailed information for the assessment of tissue ingrowth rates into porous biomaterials. This is achieved by examining a number of computer-defined concentric zones, which are based on the distance of a pixel from the scaffold edge. Each zone is automatically analyzed for tissue content, eliminating the need for user definition of a tissue ingrowth front and thus reducing errors and observer dependence. Tissue ingrowth rates in two biodegradable polyurethane scaffolds (Estane and polycaprolactone-polyurethane [PCLPU]) specifically designed for tissue engineering of the knee meniscus were assessed. Samples were subcutaneously implanted in rats with follow-up until 6 months. Especially at the earlier follow-up points, PCLPU scaffolds showed significantly higher tissue ingrowth rates than Estane scaffolds, making the PCLPU scaffold a promising candidate for further studies investigating meniscus tissue engineering.

Uncatalyzed synthesis, thermal and mechanical properties of polyurethanes based on poly(epsilon-caprolactone) and 1,4-butane diisocyanate with uniform hard segment.

Polyurethanes based on poly(epsilon-caprolactone) (PCL) (750-2800 g/mol) and 1,4-butane diisocyanate (BDI) with different soft segment lengths and constant uniform hard segment length were synthesized in absence of catalysts for the production of a degradable meniscus scaffold. First the polyesterdiols were endcapped with BDI yielding a macrodiisocyanate with a minimal amount of side reactions and a functionality of 2.0. Subsequently, the macrodiisocyanates were extended with 1,4-butanediol in order to obtain the corresponding polyurethane. The polyurethanes had molecular weights between 78 and 160 kg/mol. Above molar masses of 1900 g/mol of the polyesterdiol crystalline PCL was found while the hard segment showed an increase in melting point from 78 to 122 degrees C with increasing hard segment content. It was estimated that the percentage crystallinity of the hard segment varied between 92 and 26%. The Young's modulus varied between 30 and 264 MPa, the strain at break varied between 870 and 1200% and tear strengths varied between 97 and 237 kJ/m2.