Carl A. Kirker-Head

In vivo degradation of three-dimensional silk fibroin scaffolds

Three-dimensional porous scaffolds prepared from regenerated silk fibroin using either an all-aqueous process or a process involving an organic solvent, hexafluoroisopropanol (HFIP), have shown promise in cell culture and tissue engineering applications. However, their biocompatibility and in vivo degradation have not been fully established. The present study was conducted to systematically investigate how processing method (aqueous vs. organic solvent) and processing variables (silk fibroin concentration and pore size) affect the short-term (up to 2 months) and long-term (up to 1 year) in vivo behavior of the protein scaffolds in both nude and Lewis rats. The samples were analyzed by histology for scaffold morphological changes and tissue ingrowth, and by real-time RT-PCR and immunohistochemistry for immune responses. Throughout the period of implantation, all scaffolds were well tolerated by the host animals and immune responses to the implants were mild. Most scaffolds prepared from the all-aqueous process degraded to completion between 2 and 6 months, while those prepared from organic solvent (hexafluoroisopropanol (HFIP)) process persisted beyond 1 year. Due to widespread cellular invasion throughout the scaffold, the degradation of aqueous-derived scaffolds appears to be more homogeneous than that of HFIP-derived scaffolds. In general and especially for the HFIP-derived scaffolds, a higher original silk fibroin concentration (e.g. 17%) and smaller pore size (e.g. 100-200microm) resulted in lower levels of tissue ingrowth and slower degradation. These results demonstrate that the in vivo behavior of the three-dimensional silk fibroin scaffolds is related to the morphological and structural features that resulted from different scaffold preparation processes. The insights gained in this study can serve as a guide for processing scenarios to match desired morphological and structural features and degradation time with tissue-specific applications.

Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein.

A middiaphyseal, 2.5-cm osteoperiosteal segmental defect stabilized by plate fixation was created in the right femur of 17 sheep. Four treatment groups were included: Group I, no implant; Group II, inactive bone matrix; Group III, recombinant human bone morphogenetic protein (rhBMP-2) mixed with inactive bone matrix; and Group IV, autogeneic bone graft. Three animals had early failure of fixation, and the remaining 14 were evaluated at three months after implantation. Radiographs showed bony union of all defects treated with rhBMP-2 (six) and a lack of bony union in the negative-control groups treated with no implant (three) and inactive bone matrix without BMP (three). Both defects treated with autograft healed. New bone formation in the defect sites treated with rhBMP-2 first appeared one month after implantation and had a mean bending strength (expressed as a percentage of the contralateral femur) of 91% +/- 59% (mean +/- standard deviation) for defects treated with BMP-2, 77% +/- 34% for autograft, 9% +/- 8% for no implant, and 11% +/- 7% for inactive matrix without BMP. Three sheep treated with rhBMP-2 had their fixation plates removed at four months and were followed for one year. Their bone defect sites remained solidly healed one year after the initial operation.

Potential applications and delivery strategies for bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs) are multifunctional cytokines which are members of the Transforming Growth Factor-beta superfamily. They are the only signaling molecules which can singly induce de novo bone formation at orthotopic and heterotopic sites and their osteoinductive potency makes them clinically valuable as alternatives to bone graft. Several means of delivering BMPs to patients are undergoing evaluation including systemic administration, gene transfer and local matrix delivery vehicle implantation. The latter methodology is in advanced stages of development for application in humans in the treatment of selected spinal fusions, fracture repairs, craniomaxillofacial surgery and periodontal injury and disease. The BMPs are also widely distributed in non-skeletal tissues such as nerve, gastrointestinal tract, kidney, heart and lungs and they have a central role in vertebrate and non-vertebrate organogenesis. Initial studies indicate that the BMPs have neuro, cardio and reno-protective actions and it is likely that therapeutic indications for their use will extend well beyond skeletal disease and injury.

Healing bone using recombinant human bone morphogenetic protein 2 and copolymer.

Middiaphyseal 2.5-cm segmental defects in the right femurs of 12 sheep were stabilized with stainless steel plates and implanted with (1) 2 mg recombinant human bone morphogenetic protein 2 and poly[D,L-(lactide-co-glycolide)] bioerodible polymer with autologous blood (n= 7), (2) 4 mg recombinant human bone morphogenetic protein 2 and poly[D,L-(lactide-co-glycolide)] and blood (n = 3), or (3) poly[D,L-(lactide-co-glycolide)] and blood only (n = 2). Bone healing was evaluated for 1 year using clinical, radiographic, gross pathologic, and histologic techniques. Union occurred in three sheep in Group 1, two in Group 2, and none in Group 3. In the animals that healed, new bone first was visible radiographically between Weeks 2 and 6 after implantation; new bone mineral content equaled that of the intact femur not surgically treated by Week 16; recanalization of the medullary cavity approached completion at Week 52; and at necropsy the surgical treated femurs were rigidly healed, the poly[D,L-(lactide-co-glycolide)] was resorbed completely, and women and lamellar bone bridged the defect site. In two Group 1 sheep euthanized at Weeks 2 and 6, polymer particles were permeated by occasional multinucleated giant cells. Some plasma cells, lymphocytes, and neutrophils were present locally. The poly[D,L-(lactide-co-glycolide)] tended to fragment during surgical implantation. Despite these observations, the recombinant human bone morphogenetic protein 2/poly[D,L-(lactide-co-glycolide)] implant was able to heal large segmental bone defects in this demanding model.

Critically sized osteo-periosteal femoral defects: a dog model.

A 21-mm defect was created in 1 femoral diaphysis each of 15 dogs. Periosteum as well as a cylinder of bone was removed, and the defect was stabilized with a bone plate. Twelve of the defects were filled with an equal volume of autogenous cancellous bone harvested from the ipsilateral ilium. Three defects were left untreated. Cranial to caudal radiographs were taken postoperatively and every 4 weeks for 16 weeks. The radiographs were evaluated for healing using two ordinal scales. At 16 weeks, the dogs were euthanized and the femurs harvested for biomechanical testing and histologic evaluation. Both operated and contralateral not operated femurs were mechanically tested to failure in torsion, and load at failure and stiffness were calculated. All dogs tolerated the procedure well, and were using the operated limb within 1 or 2 days postoperatively. There were no complications noted during the 16 weeks of the study. Unfilled defects did not heal and became atrophic nonunions. The defects filled with autogenous cancellous bone healed in a consistent pattern of consolidation, incorporation, and remodeling, with uniform increases of both ordinal scales used. The femoral cortex opposite the bone plate demonstrated most mature remodeling, evident both radiographically as well as histologically. Unoperated femurs failed at 13.61 +/- 3.88 N-m and grafted femurs failed at 2.96 +/- 1.3 N-m, which was 23% of the measurement of the unoperated femur. Relative stiffness of the unoperated femurs was 5974 +/- 4316 N-m2/radian, and grafted femurs had a relative stiffness of 642 +/- 561 N-m2/radian, which was 10.4% of the measurement of unoperated femur. This model proved to be a critically sized defect, which when left unfilled resulted in an atrophic nonunion, and when filled with cancellous bone resulted in a consistent healing pattern.

Sustained volume retention in vivo with adipocyte and lipoaspirate seeded silk scaffolds

Current approaches to soft tissue regeneration include the use of fat grafts, natural or synthetic biomaterials as filler materials. Fat grafts and natural biomaterials resorb too quickly to maintain tissue regeneration, while synthetic materials do not degrade or regenerate tissue. Here, we present a simple approach to volume stable filling of soft tissue defects. In this study, we combined lipoaspirate with a silk protein matrix in a subcutaneous rat model. Silk biomaterials can be tailored to fit a variety of needs, and here were processed silk biomaterials into a porous sponge format to allow for tissue ingrowth while remaining mechanically robust. Over an 18 month period, the lipoaspirate seeded silk protein matrix regenerated subcutaneous adipose tissue while maintaining the original implanted volume. A silk protein matrix alone was not sufficient to regenerate adipose tissue, but yielded a fibrous tissue, although implanted volume was maintained. This work presents a significant improvement to the standard approaches to filling soft tissue defects by matching biomaterial degradation and tissue regeneration profiles.

Surgical repair of rib fractures in 14 neonatal foals: case selection, surgical technique and results.

REASONS FOR PERFORMING STUDY Fractured ribs are encountered quite frequently in newborn Thoroughbred foals, often with fatal outcome. Surgical repair of fractures therefore requires consideration as a means of reducing mortality. OBJECTIVES To evaluate the repair of rib fractures using internal fixation techniques in foals at 2 different equine hospitals following similar diagnostics and case selection. METHODS The records of 14 foals that underwent internal fixation of fracture ribs were reviewed. Subject details, clinical presentation, diagnosis, surgical technique, post operative care and complications were recorded. Follow-up information was obtained in 7 foals. RESULTS The fractured ribs were reduced and stabilised using reconstruction plate(s), self-tapping cortical screws and cerclage wire in 12 cases, Steinmann pins and cerclage wires in 1 case and both techniques in 1 case. Not every rib was reduced on each case. Surgical reduction was performed on an average of 2 ribs, range 1-3 ribs in each foal. At the time of writing, 4 foals had been sold, one age 2 years was in training and 2 others died from unrelated causes. CONCLUSIONS Our data support the use of surgical stabilisation utilising reconstruction plates, self-tapping cortical screws and cerclage wire for selected cases of thoracic trauma in neonatal foals. The use of Steinmann pins may be suboptimal due to cyclic failure, implant migration and the potential for iatrogenic internal thoracic trauma. POTENTIAL RELEVANCE Foals with existing extensive internal thoracic trauma resulting from rib fracture(s), or the potential for such trauma, previously considered to have a guarded to poor prognosis for survival, may be successfully managed with internal fixation of selected fracture sites.

Pararectal Cystotomy for Urolith Removal in Nine Horses

OBJECTIVE To report outcome of horses after standing pararectal cystotomy for removal of uroliths. STUDY DESIGN Case series. ANIMALS Horses (n=9) with cystic calculi. METHODS Medical records (December 1998-May 2007) of horses with cystic calculi that had standing pararectal cystotomy were reviewed. Signalment; urolith number, size, and type; surgical technique; sedation and analgesia protocols; intra- and postoperative complications and outcome were analyzed. RESULTS Uroliths (mean diameter, 6.37 cm; median, 6 cm; range, 3-10 cm) were removed intact without need for fragmentation. Eight (89%) horses had no complications and 1 horse (11%) developed persistent drainage from the perineal incision and incisional healing was prolonged. The complications resolved after wound revision, and although cystoscopy showed absence of uroliths, the clinical signs associated with cystitis recurred. CONCLUSIONS Cystic calculi can be removed safely in standing horses using a pararectal approach. The procedure was well tolerated and no serious complications were encountered. CLINICAL RELEVANCE Pararectal cystotomy allows removal of cystic calculi in standing sedated horses. The technique may offer an economic advantage over approaches that require general anesthesia.

Core decompression of the equine navicular bone: an in vivo study in healthy horses.

OBJECTIVE To determine the physiologic response of the equine navicular bone to core decompression surgery in healthy horses. STUDY DESIGN Experimental in vivo study. MATERIALS Healthy adult horses (n=6). METHODS Core decompression was completed by creating three 2.5-mm-diameter drill channels into the navicular bone under arthroscopic control. The venous (P(V)), arterial (P(A)), articular (P(DIPJ)), and intraosseous pressures (IOP) were recorded before and after decompression drilling. Each IOP measurement consisted of a baseline (IOP(B)) and a stress test (intramedullary injection of saline solution, IOP(S)) recording. Lameness was assessed subjectively and using force plate gait analysis. Fluorochrome bone labeling was performed. Horses were euthanatized at 12 weeks. Navicular bone mineral density (BMD) was measured, and bone histology evaluated. RESULTS Peak IOP (IOP(max)) after stress testing was significantly (P<.05) reduced immediately after core decompression; however, the magnitude of these effects was decreased at 3 and 6 weeks after decompression. A significant (P<.05) correlation existed between IOP(max) and BMD. No lameness was observed beyond the first week after surgery. Substantial remodeling and neovascularization was evident adjacent the surgery sites. CONCLUSION Navicular bone core decompression surgery reduced IOP(max), and, with the exception of a mild short-lived lameness, caused no other adverse effects in healthy horses during the 12-week study period.Objective: To determine the physiologic response of the equine navicular bone to core decompression surgery in healthy horses. Study Design: Experimental in vivo study. Materials: Healthy adult horses (n=6). Methods: Core decompression was completed by creating three 2.5-mm-diameter drill channels into the navicular bone under arthroscopic control. The venous (PV), arterial (PA), articular (PDIPJ), and intraosseous pressures (IOP) were recorded before and after decompression drilling. Each IOP measurement consisted of a baseline (IOPB) and a stress test (intramedullary injection of saline solution, IOPS) recording. Lameness was assessed subjectively and using force plate gait analysis. Fluorochrome bone labeling was performed. Horses were euthanatized at 12 weeks. Navicular bone mineral density (BMD) was measured, and bone histology evaluated. Results: Peak IOP (IOPmax) after stress testing was significantly (P<.05) reduced immediately after core decompression; however, the magnitude of these effects was decreased at 3 and 6 weeks after decompression. A significant (P<.05) correlation existed between IOPmax and BMD. No lameness was observed beyond the first week after surgery. Substantial remodeling and neovascularization was evident adjacent the surgery sites. Conclusion: Navicular bone core decompression surgery reduced IOPmax, and, with the exception of a mild short-lived lameness, caused no other adverse effects in healthy horses during the 12-week study period.

Vascular-Mineral Spatial Correlation in the Calcifying Turkey Leg Tendon

Certain avian tendons are known to mineralize normally in a gender-related manner. These tendons have unique properties, as well as several features common to calcifying cartilage and bone in other vertebrate species. In cartilage and bone, mineralization has been associated with a vascular and microvascular origin, but such a relationship has not been established in the avian tendon. Here the vasculature and mineral deposition in the calcifying leg tendon of the turkey have been documented for the first time utilizing a combination of histology, immunochemistry, radiology, and novel three-dimensional image reconstruction methods based on computed tomography. The data describe the blood supply to turkey leg tendon and show that the vascularity of this tissue is correlated with the onset of mineralization: At local levels of structural hierarchy, a vascular bed precedes mineral formation and the sites of vascular invasion correspond spatially with those of mineral deposition. Relatively unvascularized tendon regions remain uncalcified. These results support the concept that vascularization and mineralization are functionally related in the tendon, and they suggest that vascular invasion provides a mechanism underlying subsequent mineral deposition in this tissue.