Nerve regeneration using tubular scaffolds from biodegradable polyurethane.

INTRODUCTION: In severe nerve lesion, nerve defects and in brachial plexus reconstruction, autologous nerve grafting is the golden standard. Although, nerve grafting technique is the best available approach a major disadvantages exists: there is a limited source of autologous nerve grafts. This study presents data on the use of tubular scaffolds with uniaxial pore orientation from experimental biodegradable polyurethanes coated with fibrin sealant to regenerate a 8 mm resected segment of rat sciatic nerve. METHODS: Tubular scaffolds: prepared by extrusion of the polymer solution in DMF into water coagulation bath. The polymer used for the preparation of tubular scaffolds was a biodegradable polyurethane based on hexamethylene diisocyanate, poly(epsilon-caprolactone) and dianhydro-D-sorbitol. EXPERIMENTAL MODEL: Eighteen Sprague Dawley rats underwent mid-thigh sciatic nerve transection and were randomly assigned to two experimental groups with immediate repair: (1) tubular scaffold, (2) 180 degrees rotated sciatic nerve segment (control). Serial functional measurements (toe spread test, placing tests) were performed weekly from 3rd to 12th week after nerve repair. On week 12, electrophysiological assessment was performed. Sciatic nerve and scaffold/nerve grafts were harvested for histomorphometric analysis. Collagenic connective tissue, Schwann cells and axons were evaluated in the proximal nerve stump, the scaffold/nerve graft and the distal nerve stump. The implants have uniaxially-oriented pore structure with a pore size in the range of 2 micorm (the pore wall) and 75 x 700 microm (elongated pores in the implant lumen). The skin of the tubular implants was nonporous. Animals which underwent repair with tubular scaffolds of biodegradable polyurethanes coated with diluted fibrin sealant had no significant functional differences compared with the nerve graft group. Control group resulted in a trend-wise better electrophysiological recovery but did not show statistically significant differences. There was a higher level of collagenic connective tissue within the scaffold and within the distal nerve stump. Schwann cells migrated into the polyurethane scaffold. There was no statistical difference to the nerve graft group although Schwann cell counts were lower especially within the middle of the polyurethane scaffold. Axon counts showed a trend-wise decrease within the scaffold. CONCLUSION: These results suggest that biodegradable polyurethane tubular scaffolds coated with diluted fibrin sealant support peripheral nerve regeneration in a standard gap model in the rat up to 3 months. Three months after surgery no sign of degradation could be seen.

Biodegradable polyurethane cytocompatibility to fibroblasts and staphylococci.

Biodegradable polyurethanes have potential for use as implantable devices (orthopedic, maxillofacial, cardiovascular, wound dressing and plastic surgery) because of their controllable elasticity, and the possibility of changing their chemistry and structure. Studying bacterial and cell adhesion to polyurethanes helps to determine surface cytocompatibility and suitability for in vivo trials. Staphylococcus aureus, Staphylococcus epidermidis and hTERT human fibroblast cells were used to determine the cytocompatibility of experimental biodegradable polyurethanes (PUs) with different hydrophobic-to-hydrophilic (pho:phi) content ratios (100% pho, 70% pho, and 30% pho). Poly(L/DL-lactide) 70/30% (PLDL) and Thermanox were used as control surfaces. Surface characterization using noncontact profilometry, contact angles, and scanning electron microscopy (SEM) showed that the three PU surfaces, PLDL, and Thermanox have different properties. On the 100% PU and 30% PU surfaces, hTERT cells spread less in comparison to the 70% PU, PLDL, and Thermanox surfaces. The adsorption of fibronectin to the surfaces had no effect on the adhesion and spreading of hTERT cells when compared to the uncoated surfaces. The trend for S. aureus was the most adhered on the 70% PU and 30% PU, then Thermanox, followed by 100% PU and PLDL, respectively. The amount of S. epidermidis adhesion followed the trend of the most on 70% PU, then 100% PU, then 30% PU and PLDL, and the least on Thermanox. These results suggest that the 70% PU surface is cytocompatible to hTERT fibroblasts, while the 100% PU and 30% PU were not. All surfaces encouraged S. aureus and S. epidermidis colonization, particularly the 70% PU.

[Reconstruction of the orbits with polylactate implants: animal experimental results after 12 months and clinical prospects]. / Rekonstruktion der Orbita mit Polylaktat-Implantaten: tierexperimentelle Ergebnisse nach 12 Monaten und klinischer Ausblick.

In earlier experiments healing of large orbital wall defects in sheep occurred undisturbed by osteoconductive bone growth along biodegradable membranes when there was no interference with additional bone grafts or titanium miniplate osteosynthesis. In this experiment similar bilateral defects were reconstructed with poly(L/DL 80/20) lactide implants using a microporous membrane 0.5 mm thick without further support on one side, an 0.25 mm microporous membrane supported by solid polylactide buttresses and stabilized by polylactide dowels on the opposite side. After 12 months we found a symmetrical reconstruction of the normal anatomy of the orbits in CT and X-ray examinations. In contrast, histologic investigations revealed massive foreign-body reactions around degrading buttress implants and dowels especially. Milder reactions occurred in some orbits along the membranes as well, in contrast to our earlier experiments with 4-month follow-up. None of the implants had degraded completely 12 months after surgery. In our 12-month long-term survey, polylactide microporous membranes confirmed their osteoconductive potential in orbital wall reconstruction. Nevertheless, massive polylactic implants should not be considered for clinical application in the orbit because of significant late foreign-body reactions.

Bone regeneration in segmental defects with resorbable polymeric membranes: IV. Does the polymer chemical composition affect the healing process?

Diaphyseal segmental defects 10 mm in length in the radii of 36 skeletally mature rabbits were covered with tubular microporous membranes prepared from poly(L/D-lactide) (18 rabbits) and poly(L/DL-lactide) (18 rabbits) to determine whether chemical composition of the membrane affected the bone healing in the defect. The results of a previous study in which similar defects of the rabbits radii were not covered with membranes or covered with poly(L-lactide) membranes were used as controls. The control defects were rapidly filled with overlying muscle and soft tissues, producing a radio-ulnar synostosis. The osseous activity of control defects was limited to the bone ends. The defects covered with membranes were progressively filled with new bone. At 1 year, complete bone regeneration in the defects covered with the poly(L/D-lactide) membrane was found in 16 cases, no regeneration in 1 animal and pseudoarthrosis in 1 animal. For the poly(L/DL-lactide) membrane there was complete bone regeneration in 17 cases (1 animal died during surgery). The quality of the interface between the new bone and the membrane seemed to be affected by the chemical structure of the polylactides used for membranes preparation. For poly(L/D-lactide), the connective tissue layer entirely separated the new bone from the polymeric membrane. This has been observed before for poly(L-lactide) membranes. In the case of poly(L/DL-lactide) the new bone was formed in some places in direct contact with the membrane and the membrane fragments were osteointegrated. The differences in chemical composition of the polylactide membranes did not have an evident effect on the bone regeneration process in segmental defects of the rabbit radii.

Bioresorbable polymers in trauma and bone surgery.

During the last few decades interest in resorbable polymeric materials has been steadily increasing. As with other materials for implantable devices, they have to satisfy several biological and technical requirements. Implants should maintain adequate mechanical properties in vivo for the time required and degrade at an effective rate. The conditions of polymer synthesis, further processing into implants and the sterilization process determine the mechanical properties of resorbable implants. Degradation of implants is manifested by implant fragmentation, strength loss and the decrease of polymer molecular weight. The rate of degradation and the tissue reaction are strongly affected by the material chemical composition and to some extent also by the mechanical properties. Potentially, devices made from bioresorbable polymers can overcome problems associated with metal implants like stress protection, potential for corrosion, wear and debris formation, as well as the necessity of implant removal. Resorbable polymers have proven to be good materials for a range of devices in trauma surgery. However, modifications and optimizations are still required. Three-dimensional porous scaffolds in various geometrical forms offer a good potential for the manufacture of tissue-engineered implants in the future.

In vitro growth and activity of primary chondrocytes on a resorbable polylactide three-dimensional scaffold.

Sheep articular chondrocytes were cultured for 3, 6, and 9 weeks on a three-dimensional porous scaffold from poly(L/DL-lactide) 80/20%. Cell growth and activity was estimated from the amount of proteoglycans attached to the polylactide scaffold and the amounts of DNA and proteins measured in the cell lysate. Cell morphology was assessed from scanning electron microscopy. Histochemical staining of proteoglycans present in the sponge was used to visualize the chondrocyte ingrowth in the scaffold. The amounts of DNA, proteins, and proteoglycans increased with time of culturing. Chondrocytes on the polylactide scaffold maintained their round shape. The cell ingrowth into the sponge progressed with time of culturing and proceeded from the upper surface of the sponge toward its lower surface. At 9 weeks, the chondrocytes filled the whole scaffold and reached the opposite side of the sponge. The proteoglycans network was, however, more dense at the upper half of the scaffold.

Intramedullary fixation by resorbable rods in a comminuted phalangeal fracture model. A biomechanical study.

The mechanical rigidity of three different methods of resorbable intramedullary fixation (bone peg, and polyglycolide rods with and without interlocking) was assessed in a comminuted phalangeal fracture model and the results compared with two commonly used internal fixation devices (lateral plate, crossed K-wires) in a cadaver model. Each fixation technique was tested for its biomechanical strength in apex palmar bending, compression and torsion. Failure testing for the three resorbable methods was also done. The results showed that lateral plating provided the best rigidity in apex palmar bending and torsion, followed by intramedullary bone peg fixation. All resorbable intramedullary fixations had rigidity that was at least the same as crossed K-wires. For the torque test, polyglycolide rods with interlocking provided better rigidity than without interlocking. There was no significant difference between the different methods in the compression test, except that the intramedullary bone peg was significantly stiffer than K-wires.

Regeneration of segmental diaphyseal defects in sheep tibiae using resorbable polymeric membranes: a preliminary study.

OBJECTIVE: To investigate whether a long bone cortex of well-defined thickness can be regenerated by using an anatomically designed membranous resorbable "tube-in-tube" implant and to establish the functions of membranes in the healing of segmental diaphyseal bone defects larger than the "critical size." DESIGN: Bone healing in segmental diaphyseal defects larger than the critical size in the sheep tibiae covered with a single porous tubular membrane or implanted with anatomically shaped porous double tube-in-tube membranes was evaluated. Membranes with different pore structures were applied alone and/or in combination with autogenous bone graft. BACKGROUND: Healing of segmental diaphyseal bone defects in animals can be enhanced by covering the defects with resorbable polylactide membranes. Based on the results of bone healing in defects ten millimeters long in the rabbit radii, it was suggested that the membrane prevents muscle and soft tissue from invading the defect and maintains osteogenic cells and osteogenic substances within the space covered with membrane, thus promoting new bone formation. The functions of membranes may differ, however, depending on the size and the location of the defect and on the experimental species used. Bone defects larger than the critical size may not heal at all, even if membranes are used. The critical-size defect is defined as the smallest bone defect that does not heal spontaneously when covered with polymeric membranes. To heal such defects, it is mandatory that membranes are used in combination with autogenic bone graft and/or a suitable bone substitute. If bone graft is used to fill the defect, the structure and geometry of the covering membrane will determine whether the graft will be vascularized and/or nourished from the surrounding soft tissue and, in consequence, survive. It can be appreciated that bone healing in areas of good vascularity should be more efficient than bone healing in poorly vascularized areas. The influence of all these factors on healing of bone in segmental diaphyseal defects covered with membranes is not known. METHODS: Four-centimeter-long diaphyseal segmental defects in the tibiae of six- to seven-year-old Swiss mountain sheep were covered with resorbable membranes from poly(LDL-lactide). In Group 1, a single microporous external membrane was used. In Group 2, one microporous membrane was inserted into the medullary cavity at the cut ends of the tibiae (internal membrane), and the other microporous membrane was placed on the outer surface of the cortex (external membrane). In Group 3, a single microporous external membrane was also laser-perforated to produce openings with a diameter in the range of 800 to 900 micrometers. In Group 4, the defect was filled with autogenous cancellous bone graft and covered with a single perforated membrane. In Group 5, one perforated internal membrane was inserted into the medullary cavity at the cut ends of the tibiae, and the other perforated membrane was placed on the outer surface of the cortex. Group 6 was identical to Group 5, except that cancellous bone graft was placed in the space between these two membranes. RESULTS: There was no bone healing in Groups 1, 2, 3, and 5. Only in Groups 4 and 6 did the defects heal. In Group 4, new bone was dispersed across the "medullary canal" formed by the membrane. In Group 6, the new bone had grown into the space between the outer and inner membranes, forming the "neocortex." CONCLUSIONS: The resorbable polymeric implant consisting of two concentric perforated membranes (the tube-in-tube implant) used in combination with cancellous bone graft to treat segmental diaphyseal defects in sheep tibiae allows for the reconstitution of the "neocortex" with well-defined thickness. (ABSTRACT TRUNCATED)

Improvement of patency rate in heparin-coated small synthetic vascular grafts.

BACKGROUND: Graft thrombosis and intimal hyperplasia represent the major causes of graft failure. Heparin has been shown to have a beneficial effect on long-term patency and on prevention of intimal hyperplasia. Thus, the purpose of the present study was to evaluate the effect of heparin coating on patency rate and intimal hyperplasia in small synthetic vascular grafts. METHODS AND RESULTS: Two synthetic grafts (expanded polytetrafluoroethylene [ePTFE], and polyurethane) with and without heparin coating were implanted in the infrarenal aorta (diameter, 2 mm) of 40 Whistar rats. Animals survived 8 weeks after implantation. Graft patency, intimal thickness, and percentage of diameter stenosis were determined by light microscopy at the proximal respectively distal anastomosis and in the middle of the graft. Uncoated grafts showed a patency rate of 70% for ePTFE and 60% for polyurethane grafts. Heparin-coated grafts showed a patency rate of 100% for ePTFE and 90% for polyurethane grafts. Intimal hyperplasia was observed in all grafts mainly at the anastomosis site. Intimal wall thickness and percentage of stenosis were significantly more pronounced in the polyurethane than ePTFE grafts (P < 0.01). Heparin coating significantly reduced overall graft thrombosis (P < 0.05) but had no significant effect on intimal hyperplasia. CONCLUSIONS: Small grafts show a high rate of graft thrombosis and an enhanced intimal hyperplasia. ePTFE grafts show significantly less intimal hyperplasia and percentage of stenosis than polyurethane grafts. Heparin coating significantly reduced graft thrombosis but had no significant effect on intimal hyperplasia. Thus, heparin coating seems to be beneficial for graft patency, and ePTFE appears to be superior to polyurethane as graft material.

[Experimental reconstruction of the sheep orbit with biodegradable implants]. / Experimentelle Rekonstruktion der Orbita beim Schaf mit biodegradablen Implantaten.

In a complex animal model in sheep, polydioxanone (PDS) and polylactic membranes were used for the reconstruction of large orbital-wall defects. In a long-term experiment over 1 year, polylactic implants alone showed the best performance as compared with combinations involving autogenous bone grafts and titanium miniplate fixation. As soon as these polylactic implants are approved for human surgery, they will be used to solve the still challenging problem of anatomical reconstruction of large comminuted fractures of more than one orbital wall.

[Experimental reconstruction of sheep orbits using biodegradable implants]. / Experimentelle Rekonstruktion der Orbita beim Schaf mit biodegradablen Implantaten.

In a complex animal model in sheep, polydioxanone (PDS(®)) and polylactic membranes were used for the reconstruction of large orbital-wall defects. In a long-term experiment over 1 year, polylactic implants alone showed the best performance as compared with combinations involving autogenous bone grafts and titanium miniplate fixation. As soon as these polylactic implants are approved for human surgery, they will be used to solve the still challenging problem of anatomical reconstruction of large comminuted fractures of more than one orbital wall.

Effect of in vivo and in vitro degradation on molecular and mechanical properties of various low-molecular-weight polylactides.

The in vivo and in vitro degradation of low-molecular-weight poly(L-lactide), poly(L/D-lactide), and poly (L/DL-lactide) rods was investigated. The low-molecular-weight fast-degrading materials were used to accelerate the degradation process and make the test conditions more critical. In the in vivo study the rods were implanted in the soft tissue of sheep and explanted at 1, 3, 6, and 12 months. In the in vitro experiments the samples were subjected to aging at 37 degrees C in the phosphate buffer using two different modes. In the so-called pseudodynamic mode the aging buffer was regularly replaced if the pH dropped more than 0.5. In the static mode the buffer was not changed over the whole testing period of 52 weeks. The mechanical, molecular, and crystalline properties of the rods were measured and their appearance in the course of aging was evaluated using scanning electron microscopy. It was found that the changes in the mechanical properties of poly(L-lactide), poly(L/D-lactide), and poly(L/DL-lactide) samples subjected to in vitro degradation tests in both the static and pseudodynamic modes are in good approximation with data obtained from the in vivo study. The pH of the buffer solution had no evident effect on the mechanical properties or the rate of degradation as estimated from the drop in molecular weight of the aged samples. The replacement of the aging buffer to maintain a constant pH at 7.4 does not seem to be critical for the degradation of the polylactides. In vitro degradation tests can be used as a relevant procedure for predicting the in vivo functionality of implants from the polylactides used if the criteria for assessing such a functionality are the changes in mechanical properties and molecular weight.

The biological effect of natural bone mineral on bone neoformation on the rabbit skull.

The aim of this study was to evaluate the effect of deproteinized bovine bone graft material on new bone formation in a guided bone regeneration model system. In 20 rabbits, a periosteal skin flap was raised uncovering the calvaria. A form stable hemispherical dome made of poly-lactic acid (PLA) was placed onto the roughened calvaria. Prior to placement, the dome was either filled with peripheral blood alone (control group, 8 rabbits), or with blood and OsteoGraf/N-300 (test group, 12 rabbits). The wound was closed for primary healing. Morphometric assessment of 1- and 2-month undecalcified histologic specimens revealed better tissue fill in the test domes at 1 month (test 99%, control 55%) (P < 0.05) and 2 months (t, 100%; c, 82%). The fraction of the new bone within the regenerated tissue was higher in the test specimens at 1 month (t, 22%; c, 12%) (P < 0.05) and 2 months (t, 34%; c, 24%). The fraction of the entire space underneath the domes occupied by bone was higher in the test at 1 month, but higher in the controls at 2 months. The fraction of the bone substitute material in contact with bone increased from 1 month (34% +/- 14) to 2 months (45% +/- 5). The surface fraction of osteoblast layers was tendentially higher in the test at 1 month but higher in the control specimens at 2 months. In both test and control, initially woven bone was formed which underwent subsequent remodeling. Cellular degradation of the deproteinized bone graft was frequently detected. It is concluded that deproteinized bovine bone mineral has osteoconductive properties and can initially accelerate new bone formation during guided bone regeneration by increased recruitment of osteoblasts.

The significance of angiogenesis in guided bone regeneration. A case report of a rabbit experiment.

A dome-shaped bioresorbable membrane was fixed to the wounded rabbit calvaria and filled with a bioresorbable fibre conglomerate. After 4 weeks, the histologic preparation revealed an intimate spatial and temporal correlation between newly formed blood vessels and de novo extraskeletal bone formation. These observations emphasize the significance of angiogenesis in guided bone generation.

Blood-filled spaces with and without filler materials in guided bone regeneration. A comparative experimental study in the rabbit using bioresorbable membranes.

The aim of the present study was to evaluate the effect of natural deproteinized bone mineral on the temporal and spatial pattern of bone formation in a guided bone regeneration model system while using a bioresorbable membrane device. A periosteal skin flap was raised uncovering the calvaria of 20 rabbits. A stiff hemispherical dome made of polylactic acid was placed onto the roughened calvaria and anchored by screws. Prior to placement, the dome was either filled with peripheral blood (control group, 8 rabbits) or with blood and OsteoGraf/N-300 (test group, 12 rabbits). At 1 month, histologic sections revealed bone regeneration in both test and control domes to various degrees. In the test domes, bone height reached 78% (67-83) and bone volume was 11% (6-17), while in the control domes, bone height was 45% (14-67) and bone volume 6% (1-11). At 2 months, bone height was unchanged in the test group at 70% (67-83) and bone volume had only slightly increased to 16% (11-21). In the controls, height increased to 86% (60-100) and volume to 20% (9-27). Thus, in this model system, natural bone mineral fill contributed to accelerate initial bone neogenesis, while it did not contribute to increasing bone volume or bone height at later observation stages.

Positional stability of polylactide pins with various surface textures in sheep tibia.

Resorbable pins with a smooth or textured surface were produced from poly(L-lactide) P(L)LA poly(L/D-lactide) 95/5% P(L/D)LA, and poly(L/DL-lactide) 95/5% P(L/DL)LA by injection molding. The pins were implanted in sheep tibiae to establish whether the pin surface geometry and the polymer composition used for the pin preparation affect their positional stability in bone; i.e., can the pin design and its ability to swell in body fluids protect against loosening. Three of the 32 P(L)LA pins with a smooth surface loosened but none of the P(L/D)LA and P(L/DL)LA pins with a smooth surface did. This may indicate that expansion of the pin upon swelling protects against loosening, even if the pin's surface geometry is not optimal. None of the pins with a textured surface were loose, independent of the polymer used for the pin preparation. The textured surface of these pins allowed the ongrowth of new bone and hence, implant anchoring, secured the positional stability of the implant in the bone.

Effect of thermal treatment on sterility, molecular and mechanical properties of various polylactides. 2. Poly(L/D-lactide) and poly(L/DL-lactide).

Pins for orthopaedic applications injection-moulded from as-supplied poly(L/D-lactide) 95/5% and poly(L/DL-lactide) 95/5%, raw as-supplied polymers or raw methanol-extracted polymers were heat-treated for a predetermined time at 135 degrees C under moisture-free argon. The sterility, molecular weight, polydispersity, mechanical properties, and crystallinity of the heat-treated samples were evaluated. All the samples heat treated under argon were sterile after 2 h exposure to heat. The heat-treated pins and raw as-supplied polymers showed a continuous decrease in molecular weight over the entire 50 h of heating, the decrease being more substantial for poly(L/D-lactide) than poly(L/DL-lactide). Raw methanol-extracted polymers showed an increase of molecular weight after 2 h of heat treatment, followed by a gradual decrease of molecular weight up to 50 h. A drop in the bending strength, an increase in the bending moduli, and no change in the shear strength was observed for polylactide pins during the first 5 h of thermal treatment. For both the polymers, there was a progressive increase in crystallinity over time of thermal treatment, and practically no change in the melting temperature.