Enhanced peripheral nerve regeneration through a poled bioresorbable poly(lactic-co-glycolic acid) guidance channel.

In this study we investigated the effects of materials prepared with electrical poling on neurite outgrowth in vitro and nerve regeneration in vivo. Neuro-2a cells were seeded on poled and unpoled poly(lactic-co-glycolic) (PLGA) films and observed at time periods 24, 48 and 72 h post-seeding. The percentage of cells with neurites and the neurites per cell were quantified using light microscopy. At 48 and 72 h post-seeding, both the number of cells with neurites and the neurites per cell were significantly increased on the poled films compared to those on unpoled films. An established rat sciatic nerve model was used for in vivo studies to assess the effects of PLGA guides, poled for two different periods, on peripheral nerve regeneration. Guides were inserted in rats to bridge a 1.0 cm gap created in the right sciatic nerve. After four weeks, nerves regenerated through poled guides displayed a significant increase in conduction velocity and significantly increased numbers of axons across the guides, as compared to nerves regenerating through an unpoled guidance channel. Electrical poling was shown to promote neurite growth, axon regeneration and the conduction rate of the repaired nerve. We concluded that guides prepared with electrical poling enhance peripheral nerve regeneration.

Pain control via opioid analgesic-local anesthetic loaded IPNs.

Relief of chronic pain is an important clinical problem requiring special care and approaches. The present study was designed for the construction of a controlled release system for local application of analgesics (hydromorphone (HM), morphine (M), and codeine (C)) and a local anesthetic, bupivacaine (BP). An interpenetrating network (IPN) drug release system was prepared by using a biocompatible, biodegradable copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and another biocompatible but synthetic, nondegradable polymer, poly (2- hydroxyethyl methacrylate), (PHEMA). In situ release kinetics of the IPN system was first order for BP but could not be fitted to any known equation for the other drugs. Complete release from the IPNs occurred within a considerably short time (24 h for 80 % of the drugs) most probably due to the significant hydrophilicity of PHEMA. In order to slow down the release rate these IPNs were coated with PHBV. Release from these coated IPNs (cIPN) resulted in rates that could be described by Higuchi's equations. In vivo measurement of antinociceptive efficacy was carried out in rats with tail flick and paw-withdrawal tests after inducing chronic pain created by sciatic nerve ligation at the right side. Control groups received placebo implants. In vivo studies showed potent, prolonged (2-3 days) antinociception at the site of injury (right paw) for strong opioids (HM and M) and about 2 days for the weak opioid (C) and local anesthetic (BP). In all cases the release rate was found to be as important as the antinociceptive potency. The weakest opioid analgesic of those evaluated (C) had a higher first day antinociception than its stronger counterpart M, probably due to its higher initial concentration that was expected from its faster release rate in the in situ experiments.

Antihyperalgesic effect of simultaneously released hydromorphone and bupivacaine from polymer fibers in the rat chronic constriction injury model.

We aimed to evaluate the antihyperalgesic efficacy of a combination of hydromorphone (HM) and bupivacaine (BP) delivered via controlled release from a biodegradable cylindrical rod. In vivo studies were performed using a rat model of thermal hyperalgesia induced by chronic constriction injury (CCI) of the sciatic nerve with loose ligatures. Poly(lactic-co-glycolic acid) (PLGA) rods (10 mm length, 1 mm diameter) loaded with HM (5 mg per rod), BP (5 mg per rod) or no drug (placebo) were implanted subcutaneously, in single or dual pairs, adjacent to the constriction injury, immediately after nerve ligation. We evaluated the efficacy of two dose levels for each drug, alone or in combination, in attenuating thermal hyperesthesia over a period of 12 days according to a prevention protocol. Plasma levels of drugs released from the rods and also released in an in vitro simulation were evaluated. In vitro studies demonstrated that drug release is maintained for at least 10 days. HM (5 mg) alone and BP (5 mg) alone did not attenuate hyperalgesia. Their combination provided a significant increase in the paw withdrawal latency as compared to single agents or placebo. When the dose in each group was doubled, implanting four rods, significant attenuation of hyperalgesia was observed. Analyses of rods retrieved after termination of experiments (after 12 days) revealed 30% residual HM and 70% residual BP content. Prolonged delivery of HM and BP alone or in combination via locally applied PLGA rods may offer a feasible alternative to provide long-lasting analgesia.

Mechanical evaluation of a porous bone graft substitute based on poly(propylene glycol-co-fumaric acid).

A porous, resorbable polymer composite based on poly(propylene glycol-co-fumaric acid) (PPF) was mechanically evaluated in vitro for use as a bone graft substitute and fracture fixative. The test material created a dynamic system capable of initially providing mechanical integrity to bony voids and a degradative mechanism for ingrowth by native bone. The unsaturated polymer, PPF, was crosslinked in the presence of effervescent agents to yield a porous microstructure upon curing. An in vitro degradation study first assessed the temporal mechanical properties of the test material. This research was followed by an ex vivo study using a long-bone osteotomy model to characterize the mechanics of fixation. Results showed the initial compressive strength of the cross-linked PPF system was comparable to cancellous bone. The rate of strength loss was commensurate with the predicted mechanical recovery of healing bone with analogous results in a composite that comprised also 25% (by weight) autograft. Mechanical testing in the long-bone model demonstrated that PPF-based bone-graft substitute increased the flexural strength of K-wire stabilized osteotomies. These results suggest that this type of bone graft substitute may have clinical utility in the stabilization of complex tubular bone fractures.

Enhanced bioactivity of a poly(propylene fumarate) bone graft substitute by augmentation with nano-hydroxyapatite.

The bioactivity of a nano-hydroxyapatite-augmented, bioresorbable bone graft substitute made from the unsaturated polyester, poly(propylene fumarate), was analyzed by evaluating biocompatibility and osteointegration of implants placed into a rat tibial defect. Three groups of eight animals each were evaluated by grouting bone graft substitutes into 3-mm holes that were made into the anteromedial tibial metaphysis of rats. Thus, a total of 24 animals was included in this study. Two different formulations varying as to the type of hydroxyapatite were used: Group 1 - nano-hydroxyapatite, Group 2 - micron-hydroxyapatite, with a Group 3 control defect remaining unfilled. Animals of each of the three groups were sacrificed in groups of eight at postoperative week three. Histologic analysis revealed best superior biocompatibility and osteointegration of bone graft substitutes when nanohydroxyapatite was employed. At three weeks, there was more reactive new bone formation in this group when compared to the micron-hydroxyapatite group. The control group showed incomplete closure of the defect. This study suggested that nano-hydroxyapatite may improve upon the bioactivity of bone implant and repair materials. The model scaffold used in this study, poly(propylene fumarate), appeared to provide an osteoconductive pathway by which bone will grow in faster. Clinical implications of the use potential advantages of nano-hydroxyapatite on bone repair and orthopaedic implant design are discussed.

Evaluation of a porous, biodegradable biopolymer scaffold for mandibular reconstruction.

PURPOSE: Bioresorbable bone graft substitutes could eliminate disadvantages associated with the use of autografts, allografts, and other synthetic materials. The authors investigated the osteoinductive capacity of a bioresorbable bone graft substitute made from the unsaturated polyester poly(propylene glycol-co-fumaric acid) (PPF) for mandibular reconstruction in a rat model. The eventual intention is to use this material either as a stand-alone bone graft substitute or as an extender to autograft harvested from mandibular reconstruction sites. MATERIALS AND METHODS: The PPF bone graft was crosslinked in the presence of a hydroxyapatite filler and effervescent foaming agents to develop porosity in situ by generating carbon dioxide during the effervescent reaction of citric acid and sodium bicarbonate. The latter reagents are responsible for foam formation and expansion, resulting in a polymeric scaffold with pore sizes in the range of 100 to 500 microm. Twenty adult Sprague-Dawley rats had 3-mm-diameter cortical defects decorticated on the outer aspect of their left mandibular ramus using a Hall drill. Animals were divided into 2 groups of 10 animals each. Animals in group A were treated with implantation of the PPF-based bone graft substitute. Implants were applied buccally to defects on the left side. In group B animals with similar defects, the drill holes were left to heal unaided. The amount of new bone formation and the presence of an inflammatory infiltrate were evaluated at 7 weeks postoperatively. RESULTS: Histologic analysis of the healing process revealed enhanced in vivo new bone formation with the PPF bone graft substitute. These findings were corroborated by the histomorphometric analysis of new bone formation. DISCUSSION: Results of this study demonstrated biocompatibility of the porous PPF-based scaffold in a mandibular defect. CONCLUSIONS: These findings may have applicability to the further development of bone graft substitutes for oral/maxillofacial applications.

Enhanced peripheral nerve regeneration elicited by cell-mediated events delivered via a bioresorbable PLGA guide.

Using an established rat peripheral-nerve regeneration model, the authors have demonstrated enhancement of regeneration following subcutaneous priming of bioresorbable poly(lactic-co-glycolic)acid (PLGA) guides in vivo. Four weeks after nerve reconstruction, regeneration of the peripheral nerve through the cell-infiltrated guides displayed a significant increase in the total axon number and myelination status recorded in primed over unprimed guides, demonstrating the importance of cell-mediated events in the regeneration process. To define the different components enhancing nerve regeneration in this model, they have focused on identifying factors capable of eliciting Schwann-cell migration, since this has been identified as an early and necessary event in nerve regeneration. Using an in vitro migration assay, screening of a limited number of cellular and extracellular factors has demonstrated differential promotion of Schwann-cell migration. Of interest, combining fibronectin and bFGF resulted in a two-fold enhancement in Schwann-cell migration over that recorded with either alone. These results describe a rapid screening process for identifying various molecules and combinations thereof, with potential involvement in Schwann-cell migration. Coupling these findings to the use of the PLGA guide as an in vivo delivery system provides a rationale for the selection of exogenous factors to test for the enhancement of peripheral-nerve regeneration.

Quantitative measures of osteoinductivity of a porous poly(propylene fumarate) bone graft extender.

Bioresorbable bone graft substitutes could alleviate disadvantages associated with the use of autografts, allografts, and other synthetic materials. However, little is known about the minimum autograft/extender ratio for a given material at which a sufficient osteoinductive effect is still seen. Therefore, we investigated a bioresorbable bone graft substitute made from the unsaturated polyester poly(propylene fumarate), PPF, at various mixing ratios with autograft. The bone graft extender is cross-linked in the presence of a hydroxylapatite filler and effervescent foaming agents citric acid and sodium bicarbonate. The porous bone graft extender material develops porosity in vivo by generating carbon dioxide during the effervescent reaction, resulting in foam formation and expansion with respective pore sizes of 50 to 1000 microm. In an attempt to determine how much cancellous autograft bone could be extended with the poly(propylene fumarate) material and at which ratio the autograft/extender combination remained supportive of the overall structural integrity of the repairing defect site, we studied the amount of new bone formation on implantation of the materials in 3-mm holes made in the anteromedial tibial metaphysis of Sprague-Dawley rats. The extender formulation was analyzed at high autograft/extender (75% autograft/25% extender) and low autograft/extender (25% autograft/75% extender) mixing ratios and compared with negative (extender alone) and positive (autograft alone) controls. Animals from each of the formulations were killed in groups of eight at 6 weeks postoperatively. Hence, a total of 32 animals were included in this study. Histologic analysis of the healing process revealed enhanced in vivo osteoinduction with the bone graft extender regardless of the autograft loading. Histomorphometry did not show any statistically significant difference between the high and low autograft/extender ratios. All formulations maintained implant integrity and did not provoke sustained inflammatory responses. This study suggested that the presence of even a small amount of autograft within the polymer-based bone graft extender results in significant enhancement of osteoinduction. This finding has immediate applicability to the development of bone graft extender formulations for clinical use.

Healing of osteochondral osteotomies after fixation with a hydroxyapatite-buffered polylactide. A histomorphometric and radiographic study in rabbits.

The tissue response of subchondral bone to a biodegradable fixation device manufactured in the shape of a screw and made of polylactide with a hydroxyapatite buffer were implanted through the articular surface of the intercondylar portion of the distal rabbit femur. One screw was implanted per animal. The screws had a core diameter of 3.2 mm and an outer diameter of 4.5 mm. At insertion, the implants were cut flush with the articular surface. After follow-up times of 8 and 16 weeks, the specimens were examined radiographically and histomorphometrically. The intact contralateral femur served as a control for comparison. Only minimal signs of degradation of the polymer could be seen in the histologic specimens. These implant degradation sites were commonly areas of new bone formation adjacent to the screw implant. A brim of repair tissue was formed at the entrance and exit of the implant channel. The width of the repair tissue from the tissue-implant boundary towards the center of the entrance hole varied greatly between the specimens, from 80 to 750 microm. In most specimens this bridging tissue consisted of newly formed bone and undifferentiated mesenchymal tissue. Degenerative chondrocyte clustering occurred in the pre-existing cartilage within a 400 microm wide zone from the tissue-implant interface into the recipient tissues. Some new-bone formation was seen to envelop the implant in all specimens, but the fractional osteoid formation surface of the trabeculae was only significantly higher in the screw-implanted 16-week specimens, when compared to the non-operated contralateral controls. Although the bony osteotomy was invariably healed in all specimens with good implant integration, the quality and quantity of the reparative tissue of the articular cartilage near the screw hole was variable. This study showed that large polylactide implants, which are buffered with hydroxyapatite show benign tissue responses and good implant osteointegration when implanted in bone. They may be suitable for fixation of small bone fractures. However, insertion through intra-articular surfaces may require further improvement of the implant material to avoid the degenerative repair processes seen in this study.

Biomechanical analysis of biodegradable interbody fusion cages augmented With poly(propylene glycol-co-fumaric acid).

STUDY DESIGN: Three different types of biodegradable poly(L-lactide-co-D,L-lactide) cages with and without augmentation of a biodegradable poly(propylene glycol-cofumaric acid) scaffold were compared with autograft and metallic cages of the same design and size by determining the stiffness and failure load of the L4-L5 motion segment of cadaveric human spines. OBJECTIVES: To determine how these devices limit the range of motion in the lumbar spine compared with a metallic cage. If biomechanically equivalent, biodegradable spinal fusion systems ultimately could reduce local stress shielding and diminish the incidence of clinical complications, including device-related osteopenia, implant loosening, and breakage. SUMMARY OF BACKGROUND DATA: Previous studies in dogs and humans have demonstrated vertebral body osteopenia as a result of instrumented spine fusions. To the authors' knowledge, neither an in vitro nor an in vivo biomechanical analysis of a biodegradable interbody fusion system has been performed. METHODS: Forty-eight L4-L5 motion segments were isolated from 22 male and 26 female human donors with an average age of 49.6 +/- 2.7 years (range 36-55 years). Cages of similar dimensions and design, including a threaded, hollow, porous titanium BAK cage and three different BIO cages (BIO cage 1, pure polymer; BIO cage 2, polymer plus hydroxyapatite buffer; BIO cage 3, polymer plus nano-sized hydroxyapatite), produced from the same poly(L-lactide-co-D,L-lactide) polymer were tested in a comparative analysis to intact motion segment, interbody implantation of autograft, and a BIO cage augmented with an expandable biodegradable foam-scaffold fashioned from poly(propylene glycol-cofumaric acid). RESULTS: All cages were able to increase stiffness and failure load of the unstable motion segment significantly (P < 0.01). In comparison with the bone graft, the BAK cage (P < 0.01) and BIO cages 1 and 3 (P < 0.05) were able to increase stiffness and failure load. There was no significant difference between BIO cage 2 and the bone graft. Augmentation of BIO cage 1 with the foaming PPF scaffold resulted in higher stiffness and similar failure load as seen with the BAK cage. CONCLUSION: By comparison, the in vitro lumbar spinal motion segment stiffness and failure load produced by implantation of a biodegradable interbody fusion cage augmented with an expandable PPF scaffold is similar to that of the titanium BAK cage. This suggests that biodegradable anterior interbody fusion systems could be further developed for clinical applications.

Assessment of biodegradable controlled release rod systems for pain relief applications.

Control of chronic, severe pain is a difficult and important clinical problem for most patients, especially those with cancer. Although current applications are insufficient for a satisfactory solution to this problem, the rate of disease incidence is increasing worldwide, thus making the problem more apparent. Based on this fact, this study was designed with the ultimate goal of formulating a controlled release system of pain relievers, mainly opioids, for the local treatment of pain to achieve satisfactory, fast, and less side effect-related relief and to provide a better life status for chronic pain patients. Two copolymers of a biodegradable polymer poly(L-lactide-co-glycolide) (PLGA) were used to prepare an implantable rod type drug release system containing either an analgesic or anesthetic type of pain reliever. In vitro drug release kinetics of these systems were studied. It was observed that release from PLGA 85 : 15 was more zero-order than it was from PLGA 50 : 50. A zero-order release rate was obtained for codeine, hydromorphone, and bupivacaine from PLGA (85 : 15) rods. They, however, were released from PLGA (50 : 50) rods with Higuchi kinetics. The drug solubility was also influential on release rate, as shown by the zero-order morphine release from PLGA (50 : 50) rods. Scanning electron micrographs (SEMs) of the monolithic rods revealed erosion of the rods and the removal of drug crystals from the rod structure.

Porous poly(propylene fumarate) foam coating of orthotopic cortical bone grafts for improved osteoconduction.

A porous biodegradable scaffold coating for perforated and demineralized cortical bone allografts could maintain immediate structural recovery and subsequently allow normal healing and remodeling by promoting bony ingrowth and avoiding accelerated graft resorption. This new type of osteoconductive surface modification should improve allograft incorporation by promoting new bone growth throughout the biodegradable scaffold, hence encasing the graft with the recipient's own bone. We investigated the feasibility of augmenting orthotopically transplanted cortical bone grafts with osteoconductive biodegradable polymeric scaffold coatings. Five types of bone grafts were prepared: type I, untreated fresh-frozen cortical bone grafts (negative control); type II, perforated and partially demineralized cortical bone grafts without additional coating (positive control); type III, perforated and partially demineralized cortical bone coated with a low-porosity poly(propylene fumarate) (PPF) foam; type IV, perforated and partially demineralized cortical bone coated with a medium-porosity PPF foam; and type V, perforated and partially demineralized cortical bone coated with a high-porosity PPF foam. Grafts were implanted into the rat tibial diaphysis. Fixation was achieved with an intramedullary threaded K-wire. Two sets of animals were operated on. Animals were killed in groups of eight with one set being killed 12 weeks, and the other 16 weeks, postoperatively. Radiographic, histologic, and histomorphometric analyses of grafts showed that the amount of new bone forming around the foam-coated grafts was significantly higher than that in the type I control group (uncoated) or that in type II group (perforated and partially demineralized cortical bone grafts). Although all foam formulations appeared initially equally osteoconductive, histologic evaluation of medium-porosity PPF foam-based coatings appeared to result in a sustained response 16 weeks postoperatively. Significant resorption was present in perforated and partially demineralized cortical bone graft allografts, with some accompanying new bone formation occurring primarily within the laser holes. Therefore, PPF foam-coated cortical bone grafts appeared to be better protected from excessive bone resorption, as frequently seen with invasion of fibrovascular tissue. Biomechanical analysis of the PPF foam-coated grafts corroborated findings of the morphometric analysis in that the failure strength at the allograft-host bone junction sites of all PPF-coated cortical bone grafts was higher than in the uncoated controls.

Composite resorbable polymer/hydroxylapatite composite screws for fixation of osteochondral osteotomies.

The aim of this study was to evaluate biomechanically the healing of an osteochondral fragment created in the distal sheep femur in response to fixation with a resorbable composite screw made of polylactide and hydroxylapatite. Pure poly(L-lactide) screws were used for comparison. At follow-up times of 4 or 8 weeks, specimens were examined with standard radiography, biomechanics, and histology. The intact contralateral femur served as a control. Only minimal signs of polymer degradation were seen in the histologic specimens. At 8 weeks, most osteotomies had healed completely and there was no difference in compressive strength and elastic modulus of cylindrical cores between the two types of biodegradable implants used. The width of the repair tissue at the tissue-implant interface was 250+/-50 micro m representing a clear transition zone of newly formed trabecular bone separating the implant from the surrounding plexiform bone. We conclude that relatively large polylactide implants, blended with hydroxyapatite, are capable of fixing an osteochondral fragment in an animal model. Biomechanical data assessing the quality of the bone formed at the osteotomy sites were found to be equivalent when compared to the control poly(L-lactide) implants of similar design and size. In addition, hydroxylapatite composite implants showed benign tissue responses and good implant osteointegration. Results suggest that hydroxylapatite composite screw implants can be used for similar indications as pure poly(L-lactide) implants in current clinical use.

Composite poly(lactide)/hydroxylapatite screws for fixation of osteochondral osteotomies. A morphometric, histologic and radiographic study in sheep.

The aim of this study was to evaluate the healing of an osteochondral fragment created in the distal sheep femur in response to fixation with a biodegradable polylactide/hydroxylapatite composite screw. Poly(L-lactide) screws were used for comparison. At follow-up times of 4 and 8 weeks, the specimens were examined with standard radiography and computed tomography, as well as with macro- and micro-histomorphometry. The intact contralateral femur served as a control. Only minimal signs of degradation of the polymer could be seen in the histologic specimens. At 8 weeks, nearly all osteotomies had healed completely and an association between implant type and delayed osteotomy healing was found. The width of the repair tissue at the tissue-implant interface was 250 +/- 48 microm, representing a clear transition zone of newly formed trabecular bone separating the implant from the surrounding plexiform bone. This study showed that large polylactide implants which are buffered with hydroxylapatite show benign tissue responses and good implant osteointegration. The osteotomy healing in a weight-bearing osteochondral fragment model in sheep utilizing a composite polylactide/hydroxylapatite screw was equivalent to a similar polylactide screw implant, indicating that hydroxylapatite-buffered screw implants could be used for similar indications in current clinical use.