Poly (D,L-lactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete a bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord.

Freeze-dried poly(D,L-lactic acid) macroporous scaffold filled with a fibrin solution containing Schwann cells (SCs) lentivirally transduced to produce and secrete D15A, a bi-functional neurotrophin with brain-derived neurotrophic factor and neurotrophin-3 activity, and to express green fluorescent protein (GFP) were implanted in the completely transected adult rat thoracic spinal cord. Control rats were similarly injured and then implanted with scaffolds containing the fibrin solution with SCs lentivirally transduced to produce express GFP only or with the fibrin solution only. Transgene production and biological activity in vitro, SC survival within the scaffold in vitro and in vivo, scaffold integration, axonal regeneration and myelination, and hind limb motor function were analyzed at 1, 2, and 6 weeks after implantation. In vitro, lentivirally transduced SCs produced 87.5 ng/24 h/10(6) cells of D15A as measured by neurotrophin-3 activity in ELISA. The secreted D15A was biologically active as evidenced by its promotion of neurite outgrowth of dorsal root ganglion neurons in culture. In vitro, SCs expressing GFP were present in the scaffolds for up to 6 h, the end of a typical surgery session. Implantation of SC-seeded scaffolds caused modest loss of spinal nervous tissue. Reactive astrocytes and chondroitin sulfate glycosaminoglycans were present in spinal tissue adjacent to the scaffold. Vascularization of the scaffold was ongoing at 1 week post-implantation. There were no apparent differences in scaffold integration and blood vessel formation between groups. A decreasing number of implanted (GFP-positive) SCs were found within the scaffold during the first 3 days after implantation. Apoptosis was identified as one of the mechanisms of cell death. At 1 week and later time points after implantation, few of the implanted SCs were present in the scaffold. Neurofilament-positive axons were found in the scaffold. At 6 weeks post-grafting, myelinated axons were observed within and at the external surface of the scaffold. Axons did not grow from the scaffold into the caudal cord. All groups demonstrated a similar improvement of hind limb motor function. Our findings demonstrated that few seeded SCs survived in vivo, which could account for the modest axonal regeneration response into and across the scaffold. For the development of SC-seeded macroporous scaffolds that effectively promote axonal regeneration in the injured spinal cord, the survival and/or total number of SCs in the scaffold needs to be improved.

Limitations in transplantation of astroglia-biomatrix bridges to stimulate corticospinal axon regrowth across large spinal lesion gaps.

Regrowth of injured axons across rather small spinal cord lesion gaps and subsequent functional recovery has been obtained after many interventions. Long-distance regeneration of injured axons across clinically relevant large spinal lesion gaps is relatively unexplored. Here, we aimed at stimulating long-distance regrowth of the injured corticospinal (CS) tract. During development, an oriented framework of immature astrocytes is important for correct CS axon outgrowth. Furthermore, a continuous growth promoting substrate may be needed to maintain a CS axon regrowth response across relatively large spinal lesion gaps. Hence, we acutely transplanted poly(D,L)-lactide matrices, which after seeded with immature astrocytes render aligned astrocyte-biomatrix complexes (R. Deumens, et al. Alignment of glial cells stimulates directional neurite growth of CNS neurons in vitro. Neuroscience 125 (3) (2004) 591-604), into 2-mm long dorsal hemisection lesion gaps. In order to create a growth promoting continuum, astrocyte suspensions were also injected rostral and caudal to the lesion gap. During 2 months, locomotion was continuously monitored. Histological analysis showed that astrocytes injected into host spinal tissue survived, but did not migrate. None of the astrocytes on the biomatrices survived within the lesion gap. BDA-labeled CS axons did not penetrate the graft. However, directly rostral to the lesion gap, 120.9+/-38.5% of the BDA-labeled CS axons were present in contrast to 12.8+/-3.9% in untreated control animals. The observed anatomical changes were not accompanied by locomotor improvements as analyzed with the BBB and CatWalk. We conclude that although multifactorial strategies may be needed to stimulate long-distance CS axon regrowth, future studies should focus on enhancing the viability of cell/biomatrix complexes within large spinal lesion gaps.

Surface modification of metallic cardiovascular stents by strongly adhering aliphatic polyester coatings.

This article reports on a novel two-step strategy for the coating of cardiovascular stents by strongly adhering biocompatible and biodegradable aliphatic polyesters. First, a precoating of poly(ethylacrylate) (PEA) was electrografted onto the metallic substrate by cathodic reduction of the parent monomer in dimethylformamide (DMF). The electrodeposition of PEA, in a good solvent of it, was confirmed by both Infra-red and Raman spectroscopies. The pendant ester groups of PEA were then chemically reduced into aluminum alkoxides, able to initiate the ring-opening polymerization (ROP) of either D,L-lactide (LA) or epsilon-caprolactone (CL). Growth of biodegradable PLA or PCL coatings from the adhering precoating was confirmed by both Infra-red and Raman spectroscopies, and directly observed by scanning electron microscopy (SEM). This type of coating can act as an anchoring layer for the subsequent casting of drug-loaded polyester films allowing the controlled release of antiproliferative agents for the treatment of in-stent restenosis.

In vitro and in vivo analysis of macroporous biodegradable poly(D,L-lactide-co-glycolide) scaffolds containing bioactive glass.

Recent studies have demonstrated the angiogenic potential of 45S5 Bioglass. However, it is not known whether the angiogenic properties of Bioglass remain when the bioactive glass particles are incorporated into polymer composites. The objectives of the current study were to investigate the angiogenic properties of 45S5 Bioglass particles incorporated into biodegradable polymer composites. In vitro studies demonstrated that fibroblasts cultured on discs consisting of specific quantities of Bioglass particles mixed into poly(D,L-lactide-co-glycolide) secreted significantly increased quantities of vascular endothelial growth factor. The optimal quantity of Bioglass particles determined from the in vitro experiments was incorporated into three-dimensional macroporous poly(D,L-lactide-co-glycolide) foam scaffolds. The foam scaffolds were fabricated using either compression molding or thermally induced phase separation processes. The foams were implanted subcutaneously into mice for periods of up to 6 weeks. Histological assessment was used to determine the area of granulation tissue around the foams, and the number of blood vessels within the granulation tissue was counted. The presence of Bioglass particles in the foams produced a sustained increase in the area of granulation tissue surrounding the foams. The number of blood vessels surrounding the neat foams was reduced after 2 weeks of implantation; however, compression-molded foams containing Bioglass after 4 and 6 weeks of implantation had significantly more blood vessels surrounding the foams compared with foams containing no Bioglass at the same time points. These results indicate that composite polymer foam scaffolds containing Bioglass particles retain granulation tissue and blood vessels surrounding the implanted foams. The use of this polymer composite for tissue engineering scaffolds might provide a novel approach for ensuring adequate vascular supply to the implanted device.

Characterisation of bioactive and resorbable polylactide/Bioglass composites by FTIR spectroscopic imaging.

Formation, size and distribution of hydroxyapatite domains in resorbable composites made of poly(DL-lactide) foams and Bioglass particles after exposure to a solution of phosphate-buffer saline (PBS) for different periods of time have been analysed with FTIR imaging using the micro-ATR-IR approach. The spectral information of 4096 spectra measured simultaneously with the IR microscope equipped with a focal plane infrared array detector allowed us to obtain chemical images showing the distribution of Bioglass particles in the composites. FTIR imaging in micro-ATR mode allowed to obtain images with enhanced spatial resolution. A random distribution of hydroxyapatite domains with average size of ca. 10 microm on the surface of the composites was found after exposure to PBS for 14 and 28 days. The further growth of the hydroxyapatite domains after exposure to PBS for 63 days was detected. The spectroscopic imaging method introduced here promises to become a powerful method for characterisation of resorbable polymer composites containing bioactive inorganic phases developed for bone tissue engineering scaffolds. The accurate detection of hydroxyapatite domains and the imaging of their location in the scaffold structure is required to provide an assessment of the composites bioactivity.

PDLLA/Bioglass composites for soft-tissue and hard-tissue engineering: an in vitro cell biology assessment.

The aim of this study was to examine the effect of increased content of 45S5 Bioglass (0-40 wt%) in poly(dl-lactic acid) (PDLLA) porous foams on the behaviour of MG-63 (human osteosarcoma cell line) and A549 cells (human lung carcinoma cell line). The ability of these cell lines to grow on bioactive composites was quantitatively investigated in order to assess the potentiality for their use in hard and soft-tissue engineering. Two hours after cell seeding, an increase of cell adhesion according to the increased content of Bioglass((R)) present in the foams for both cell types was observed. Cell proliferation studies performed over a period of 4 weeks showed a better aptitude of the A549 cells to proliferate on PDLLA foams containing 5 wt% Bioglass when compared to the proliferation on foams with 40 wt% Bioglass. A lower proliferation rate was obtained for cells on pure PDLLA. Scanning electron microscopy analysis showed for both cell types the presence of cells inside the porous structure of the foams. These results confirmed the biocompatibility of PDLLA/Bioglass composite foams and the positive effect of Bioglass on MG-63 cell behaviour and also showed for the first time the possibility for human lung epithelial type II cells to adhere and proliferate on these porous scaffolds. In addition, we describe a positive effect of 45S5 Bioglass on A549 cell behaviour in a dose-dependent manner, indicating the potential of using PDLLA/Bioglass composites with an optimal concentration of 45S5 Bioglass not only in bone tissue engineering but also in lung tissue engineering.

Freeze-dried poly(D,L-lactic acid) macroporous guidance scaffolds impregnated with brain-derived neurotrophic factor in the transected adult rat thoracic spinal cord.

The effects of poly(D,L-lactic acid) macroporous guidance scaffolds (foams) with or without brain-derived neurotrophic factor (BDNF) on tissue sparing, neuronal survival, axonal regeneration, and behavioral improvements of the hindlimbs following implantation in the transected adult rat thoracic spinal cord were studied. The foams were embedded in fibrin glue containing acidic-fibroblast growth factor. One group of animals received fibrin glue with acidic-fibroblast growth factor only. The foams were prepared by a thermally induced polymer-solvent phase separation process and contained longitudinally oriented macropores connected to each other by a network of micropores. Both foams and fibrin only resulted in a similar gliotic and inflammatory response in the cord-implant interfaces. With BDNF foam, up to 20% more NeuN-positive cells in the spinal nervous tissue close to the rostral but not caudal spinal cord-implant interface survived than with control foam or fibrin only at 4 and 8 weeks after implantation. Semithin plastic sections and electron microcopy revealed that cells and axons more rapidly invaded BDNF foam than control foam. Also, BDNF foam contained almost twice as many blood vessels than control foam at 8 weeks after implantation. Tissue sparing was similar in all three implantation paradigms; approximately 42% of tissue was spared in the rostral cord and approximately 37% in the caudal cord at 8 weeks post grafting. The number of myelinated and unmyelinated axons was low and not different between the two types of foams. Many more axons were found in the fibrin only graft. Serotonergic axons were not found in any of the implants and none of the axons regenerated into the caudal spinal cord. The behavioral improvements in the hindlimbs were similar in all groups. These findings indicated that foam is well tolerated within the injured spinal cord and that the addition of BDNF promotes cell survival and angiogenesis. However, the overall axonal regeneration response is low. Future research should explore the use of poly(D,L-lactic acid) foams, with or without axonal growth-promoting factors, seeded with Schwann cells to enhance the axonal regeneration and myelination response.

Tissue engineering of biphasic cartilage constructs using various biodegradable scaffolds: an in vitro study.

Biological restoration of osteochondral defects requires suitable subchondral support material that also allows the induction of hyaline cartilage tissue. Biphasic implants consisting of pre-fabricated neocartilage and an underlying biodegradable osteoconductive base may meet these requirements. Here we explore various candidate biodegradable support materials onto which neo-cartilage was produced in vitro. Porcine chondrocytes were seeded in a closed and static bioreactor with a base of biomaterial consisting of either poly-L-lactide [P(L)LA], poly-d,l-lactide [P(D,L)LA] or Collagen-hydroxyapatite [Col-HA] and were cultured for 15 weeks. Viable neo-cartilage was produced on each biomaterial with differing amounts of cellular colonisation. P(D,L)LA breakdown was more rapid and uneven among the three biomaterials, leading to constructs of irregular shape. Little or no breakdown or chondrocyte colonisation was evident in P(L)LA. Col-HA constructs were superior in terms of viability, implant morphology and integration between neo-cartilage and biomaterial. These results indicate that our reported system has potential for producing biphasic implants that may be adequate for the repair of osteochondral defects.

Evaluation of human bone marrow stromal cell growth on biodegradable polymer/bioglass composites.

Bone tissue engineering using human bone marrow mesenchymal stem cells (HBMCs) and biocompatible materials provides an attractive approach to regenerate bone tissue to meet the major clinical need. The aim of this study was to examine the effects of novel porous biodegradable composite materials consisting of a bioactive phase (45S5 Bioglass, 0, 5, and 40 wt%) incorporated within a biodegradable poly(dl-lactic acid) matrix, on HBMCs growth. Cell adhesion, spreading, and viability was examined using Cell Tracker Green/Ethidium Homodimer-1. Bone formation was assessed using scaffolds seeded with stro-1 positive HBMCs in nude mice. In vitro biochemistry indicated that with minimal scaffold pre-treatment osteoblast activity falls with increasing Bioglass content. However, 24h scaffold pre-treatment with serum resulted in a significant increase in alkaline phosphatase specific activity in 5 wt% Bioglass composites relative to the 0 and 40 wt% Bioglass groups. In vivo studies indicate significant new bone formation throughout all the scaffolds, as evidenced by immunohistochemistry.

In vivo characterisation of a novel bioresorbable poly(lactide-co-glycolide) tubular foam scaffold for tissue engineering applications.

Polylactide-co-glycolide (PLGA) foams of tubular shape were assessed for their use as soft-tissue engineering scaffolds in vitro and in vivo. Porous membranes were fabricated by a thermally induced phase separation process of PLGA solutions in dimethylcarbonate. The parameters investigated were the PLGA concentration and the casting volume of solution. Membranes produced from 5 wt/v % polymer solutions and a 6 ml casting volume of polymer solution were selected for fabricating tubes of 3 mm diameter, 20 mm length and a nominal wall thickness of 1.5 mm. Scanning electron microscopy revealed that the structure of the tubular foams consisted of radially oriented and highly interconnected pores with a large size distribution (50-300 microm). Selected tubes were implanted subcutaneously into adult male Lewis rats. Although the lumen of the tubes collapsed within one week of implantation, histological examination of the implanted scaffolds revealed that the foam tubes were well tolerated. Cellular infiltration into the foams, consisting mainly of fibrovascular tissue, was evident after two weeks and complete within eight weeks of implantation. The polymer was still evident in the scaffolds after eight weeks of implantation. The results from this study demonstrate that the PLGA tubular foams may be useful as soft-tissue engineering scaffolds with modification holding promise for the regeneration of tissues requiring a tubular shape scaffold such as intestine.