Bone Healing In Dogs Research Articles

Evaluation of β-tricalcium Phosphate (β-TCP) and Platelet Rich Plasma (PRP) Combination for Fracture Healing in Dogs Stabilized with Intramedullary Pinning

Background: The use of synthetic bone substitute,b-tricalcium phosphate (β-TCP) as an osteo-conductive material and platelet-rich plasma (PRP) as an osteo-inductive material is the alternate strategies for replenishing bone loss. Thus, the present study was conducted to evaluate the bone healing efficacy of β-TCP and PRP combination in dogs with long bone fracture repaired with threaded intramedullary pinning. Methods: Fifteen dogs were selected, which were allotted to two groups irrespective of age, breed and sex. In six dogs of Group I intramedullary pinning was performed, while in nine dogs of Group II β-TCP and PRP was applied at fracture site after stabilising with intramedullary pin. Clinical and radiographic examinations were conducted preoperatively and postoperatively on 15th, 30th and 60th day. Result: The mean weight bearing and radiographic union scores were better in group II animals as compared to group I at different time intervals. The present findings suggest that β-TCP and PRP effectively enhances the bone healing in dogs with long bone fracture.

Application of modified porcine xenograft by collagen coating in the veterinary field: pre-clinical and clinical evaluations.

This study aimed to identify a collagen-coating method that does not affect the physicochemical properties of bone graft material. Based on this, we developed a collagen-coated porcine xenograft and applied it to dogs to validate its effectiveness. Xenografts and collagen were derived from porcine, and the collagen coating was performed through N-ethyl-N'-(3- (dimethylamino)propyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) activation. The physicochemical characteristics of the developed bone graft material were verified through field emission scanning electron microscope (FE-SEM), brunauer emmett teller (BET), attenuated total reflectance-fourier transform infrared (ATR-FTIR), and water absorption test. Subsequently, the biocompatibility and bone healing effects were assessed using a rat calvarial defect model. The physicochemical test results confirmed that collagen coating increased bone graft materials' surface roughness and fluid absorption but did not affect their porous structure. In vivo evaluations revealed that collagen coating had no adverse impact on the bone healing effect of bone graft materials. After confirming the biocompatibility and effectiveness, we applied the bone graft materials in two orthopedic cases and one dental case. Notably, successful fracture healing was observed in both orthopedic cases. In the dental case, successful bone regeneration was achieved without any loss of alveolar bone. This study demonstrated that porcine bone graft material promotes bone healing in dogs with its hemostatic and cohesive effects resulting from the collagen coating. Bone graft materials with enhanced biocompatibility through collagen coating are expected to be widely used in veterinary clinical practice.

Effects of Autologous Microfragmented Adipose Tissue on Healing of Tibial Plateau Levelling Osteotomies in Dogs: A Prospective Clinical Trial.

The aim of this study was to evaluate the effects of autologous microfragmented adipose tissue (MFAT) applied after mechanical fragmentation and assess these effects radiographically in bone healing in dogs subjected to tibial plateau levelling osteotomy (TPLO). Twenty dogs with unilateral cranial cruciate ligament disease were enrolled and randomly assigned to the treatment group (MFAT) or the control group (NT). The MFAT group underwent TPLO and autologous MFAT intra-articular administration, while the NT group underwent TPLO alone. Adipose tissue was collected from the thigh region, and MFAT was obtained by mechanical fragmentation at the end of the surgery. The patients were subjected to X-ray examination preoperatively, immediately postoperatively (T0), and at 4 (T1) and 8 (T2) weeks postoperatively. Two radiographic scores that had previously been described for the evaluation of bone healing after TPLO were used. A 12-point scoring system (from 0 = no healing to 12 = complete remodelling) was used at T0, T1, and T2, while a 5-point scoring system (from 0 = no healing to 4 = 76-100% of healing) was used at T1 and T2. The median healing scores were significantly higher at T1 and T2 for the MFAT group compared with the NT group for the 12-point (p < 0.05) and 5-point (p < 0.05) scoring systems. The intra-articular injection of autologous microfragmented adipose tissue can accelerate bone healing after TPLO without complications.

The role of VEGF and BMP-2 in stimulation of bone healing with using hybrid bio-composite scaffolds coated implants in animal model

BackgroundOsteogenesis and angiogenesis are two closely correlated processes during bone growth, development, remodeling, and repair. Vascular endothelial growth factor (VEGF) is an essential mediator during the process of angiogenesis. The bone morphogenetic protein (BMP-2) family of growth factors plays critical roles in bone formation. VEGF has the potential to enhance BMPs-induced bone formation.PurposeThis study attempted to assess VEGF and BMP-2 reflecting the effect of hybrid bio-composite scaffold on bone healing in dogs and evaluate the quality of the healing process radiologically.MethodsThis study was conducted on 12 adult mongrel dogs. All dogs were divided into four equal groups (n = 3 each): chitosan non-medicated (CH) (NM), chitosan medicated (CH) (M), chitosan bioglass non-medicated (CH.BG) (NM), and Chitosan Bioglass Medicated (CH.BG) (M). VEGF and BMP-2 were evaluated during fracture healing.ResultsResults have showed a non-significant decrease in serum VEGF activity in the (CH.BG) (M) group when compared to other groups during 2, 3 weeks, followed by gradual decrease, then increase at 12 weeks of interval period. There was highly significant increase from pre-surgery to 12 weeks in serum BMP-2 levels in the (CH.BG) (M) group when compared to other groups.ConclusionBiochemical parameters along with clinical and radiographical provide sound knowledge on the degree of bone healing with the use of chitosan bio-glass medicated by risedronate sodium drug. The statistical analysis will include the Fisher exact test and T test with significance level P < 0.05 (AU).Graphical abstract

The effect of lysophosphatidic acid using a hydrogel or collagen sponge carrier on bone healing in dogs.

The purposes of this study were to determine: 1) the efficacy of polycaprolactone-g-polyethylene glycol (PCL-g-PEG) and polylactic-co-glycolic acid (PLGA-g-PEG) hydrogels and an absorbable collagen sponge (ACS) as carriers for lysophosphatidic acid (LPA), 2) the effect of LPA on bone healing in dogs, and 3) the ideal dose of LPA to maximally stimulate bone healing. Bilateral ulnar ostectomies were performed on purpose bred dogs. Control defects were filled with a PCL-g-PEG or PLGA-g-PEG hydrogel, or a saline soaked ACS. Contralateral defects were filled with a PCL-g-PEG or PLGA-g-PEG hydrogel, or an ACS with each carrying differing concentrations of an LPA solution. Dual-energy X-ray absorptiometry (DXA) was performed. Total bone area (TBA), mineral density (BMD), and mineral content (BMC) were determined at each time point. Relationships between the effect of treatment over time on TBA, BMC and BMD were determined. Phase 1 - There was no significant difference in DXA-based TBA (p = 0.09), BMC (p = 0.33), or BMD (p = 0.74) over time between LPA treatments, or between the LPA treated and control groups TBA (p = 0.95), BMC (p = 0.99), or BMD (p = 0.46). Phase 2 - There was no significant difference over time between LPA treatments in DXA-based TBA (p = 0.33), BMC (p = 0.45), or BMD (p = 0.43), or between the LPA treated and control groups TBA (p = 0.94), BMC (p = 0.38), or BMD (p = 0.17). Phase 3 - There was no significant difference over time between LPA treatments in DXA-based TBA (p = 0.78), BMC (p = 0.88), or BMD (p = 0.35), or between the LPA treated and control groups TBA (p = 0.07), BMC (p = 0.85), or BMD (p = 0.06). There was a significant increase in TBA (p <0.0001) and BMC (p = 0.0014), but a significant decrease in BMD (p <0.0001) was noted over time when all groups were combined. Although LPA has shown promise as an osteoinductive agent in research, its performance as a bone graft substitute, as utilized in this study, is unsupported. Further studies are necessary to determine the incorporation and elution kinetics of LPA from the PLGA-g-PEG hydrogel and from an ACS. Hydrogels may have clinical applications for delaying or preventing bone formation.

Use of Synthetic Bone Material with Osteoinductive Proteins to Promote Bone Healing in Dogs

Bone grafts are essential for promoting bone healing in some orthopedic cases, and synthetic bone materials have been widely used for bone defects. In addition, osteoinductive proteins such as bone morphogenetic protein and fibroblast growth factor promote osteoblast differentiation and proliferation. The combination of these factors is very useful clinically for promoting bone healing. In this study, we report the use of synthetic bone materials and osteoinductive proteins to repair bone defects in two dogs.

Scintigraphic Evaluation of Osseointegrative Response around Calcium Phosphate-Coated Titanium Implants in Tibia Bone: Effect of Platelet-Rich Plasma on Bone Healing in Dogs

Differential diagnoses for canine bone disease are commonly based on radiography. However, scintigraphy is a nuclear scanning test to trace bone healing during stress fracture and osteomyelitis and thus can be used to probe the osseointegration process in vivo in the context of the dental implant. The purpose of this study was to explore the possibility of using noninvasive scintigraphy to probe the effect of platelet-rich plasma (PRP) to enhance osseointegration of the implant and to confirm the result with a parallel histological examination in adult beagle dogs. Two independent calcium phosphate-coated titanium screw implants 8 mm in length and 4 mm in diameter were inserted into each tibia in 4 dogs (4 implants/animal, total = 16). The PRP-treated group was deposited with 0.5 ml of PRP into the drill hole in the right tibia; the control group was instilled with 0.5 ml of saline into the drill hole in the left counterpart before titanium screw implantation. Radiography of the implanted tibias showed complete osseointegration but no osteomyelitis. No difference in the ossification of the peri-implant bone on the radiographs was observed between the two groups during the experimental period. However, the scintigraphic findings revealed obvious differences in bone uptake between the two groups. The higher bone uptake in the PRP-treated group significantly increased bone activity and faster bone regeneration compared to the control group (p < 0.05) at 4 weeks after implantation. The percentage of the bone-to-implant contact on histological examination in the PRP-treated group showed dense cortical bone formation in close contact with the metallic surface of the titanium implant (p < 0.05). Quantitative histological analysis correlated well with that of the scintigraphic evaluation as revealed by Pearson correlation analysis (r = 0.72, p < 0.05). Thus, scintigraphy is a sensitive and noninvasive method suitable for osseointegration of the implant in vivo.

Healing of Extraction Sockets and Augmented Alveolar Defects Following 1-Year Treatment With Bisphosphonate

To assess the effect of bisphosphonates on healing of extraction sockets and augmented alveolar defects, 12 adult female mongrel dogs were assigned to 2 experimental groups and a control group. The experimental groups received oral alendronate (ALN, 3.5 mg/kg/wk) or IV pamidronate (PAM, 1 mg/kg/wk) for 12 months. Animals were randomly tested for serum C-terminal telopeptide of collagen I (CTx). The right first and second premolars were extracted. After 8 weeks, extraction sites were evaluated for healing. Subsequently, 3-wall defects were created in ridges and filled with human mineralized cortical particulate bone. Two months post-augmentation, animals were sacrificed and mandibles were collected for cone-beam computed tomography (CBCT) and histomorphometric appraisal. The obtained data were compared using 1-way ANOVA test. CTx test results in both experimental groups were comparable (<10 pg/mL) but lower than that of the control group (minimum 159.2 pg/mL). Two months post-extraction, bone sequestra were noticed in extraction sites in BP-treated groups, involving the entire alveolar bone in the PAM group and the upper rim of the alveoli in the ALN group. Histologically, bone sequestra from the PAM group demonstrated empty osteocyte lacunae, while in the ALN group areas of necrotic bone along with evidence of active bone remodeling was distinguished. Eight weeks post-augmentation, the experimental groups showed no evidence of bone formation in the augmented area, while bone formation ratio was measured to be 18.32% in the control group. The mean amount of pixel intensity calculated from the CBCT images of the ALN, PAM, and control group was 113.69 ± 11.04, 124.94 ± 4.72, and 113.69 ± 6.63, respectively. Pixel intensity in PAM-treated group was significantly higher than both other groups. This study demonstrated that 1-year treatment with ALN/PAM was associated with impairment of post-extraction and post-augmentation bone healing in dogs.

Effects of long-term administration of carprofen on healing of a tibial osteotomy in dogs

To evaluate effects of long-term administration of carprofen on healing of a tibial osteotomy in dogs. 12 healthy female Beagles. A mid-diaphyseal transverse osteotomy (stabilized with an intramedullary pin) of the right tibia was performed in each dog. The carprofen group (n = 6 dogs) received carprofen (2.2 mg/kg, PO, q 12 h) for 120 days; the control group (6) received no treatment. Bone healing and change in callus area were assessed radiographically over time. Dogs were euthanized 120 days after surgery, and tibiae were evaluated biomechanically and histologically. The osteotomy line was not evident in the control group on radiographs obtained 120 days after surgery. In contrast, the osteotomy line was still evident in the carprofen group. Callus area was significantly less in the carprofen group, compared with the area in the control group, at 20, 30, and 60 days after surgery. At 120 days after surgery, stiffness, elastic modulus, and flexural rigidity in the carprofen group were significantly lower than corresponding values in the control group. Furthermore, histologic evaluation revealed that the cartilage area within the callus in the carprofen group was significantly greater than that in the control group. Long-term administration of carprofen appeared to inhibit bone healing in dogs that underwent tibial osteotomy. We recommend caution for carprofen administration when treating fractures that have delays in healing associated with a reduction in osteogenesis as well as fractures associated with diseases that predispose animals to delays of osseous repair.

Non-steroidal anti-inflammatory drugs inhibit bone healing: A review

The ability of non-steroidal anti-inflammatory drugs (NSAID) to inhibit bone healing has been established in experimental animal models using mice, rats, and rabbits. The mechanism of action is largely unknown but stems from prostaglandin inhibition and is likely multifactorial. In human medicine NSAID are known to prevent heterotopic ossification, however the clinical importance of their effects on bone healing remains controversial. Although a small handful of reports suggest that NSAID suppress bone healing in dogs and horses, there is little published information to direct veterinary practice in domestic species.

Comparison of two doses of recombinant human bone morphogenetic protein in absorbable collagen sponges for bone healing in dogs

To determine the effects of 2 doses of recombinant human bone morphogenetic protein-2 in an absorbable collagen sponge (rhBMP-2/ACS) on bone healing in dogs. 27 adult dogs. Dogs underwent a mid-diaphyseal (1-mm) tibial osteotomy (stabilized with external skeletal fixation) and received an ACS containing 0.28 mg (0.2 mg/mL) or 0.56 mg (0.4 mg/mL) of rhBMP-2 or no treatment (control dogs). All dogs were examined daily; bone healing was assessed via radiography and subjective lameness evaluation every 2 weeks. After euthanasia at 8 weeks, tibiae were evaluated biomechanically and histologically. Control dogs required antimicrobial treatment for pin-site-related complications more frequently than did rhBMP-2/ACS-treated dogs. At 4 and 6 weeks, weight bearing was greater in dogs treated with rhBMP-2/ACS (0.2 mg/mL) than in control dogs, albeit not significantly. Compared with control treatment, both doses of rhBMP-2/ACS accelerated osteotomy healing at 4, 6, and 8 weeks, and the 0.2 mg/mL dose enhanced healing at 2 weeks; healing at 6 weeks was greater for the lower-dose treatment than for the higher-dose treatment. Histologically, healing at 8 weeks was significantly improved for both rhBMP-2/ACS treatments, compared with control treatment. Among groups, biomechanical variables did not differ, although less osteotomy-site failures occurred in rhBMP-2/ACS-treated groups. In dogs that underwent tibial osteotomy, rhBMP-2/ACS (0.2 mg/mL) appeared to accelerate bone healing and reduce lameness (compared with control treatment) and apparently augmented bone healing more than rhBMP-2/ACS (0.4 mg/mL). Compared with control dogs, rhBMP-2/ACS-treated dogs required antimicrobial treatments less frequently.

Effect of a static magnetic field on bone healing in the dog: radiographic and histopathological studies

Although the promotional effects on bone healing of pulsed electromagnetic fields (PEMF) have been well demonstrated, the effects of static magnetic fields (SMF) remained unclear. In this study, effects of a custom-made magnetic wrap on radiographic and histopathological aspects of bone healing using a canine unstable osteotomy gap model were investigated. After an osteotomy of the midshaft radius, bone healing was evaluated over an 8-week-period in the control dogs (n = 5) and dogs exposed to SMF (1000 gauss) (n = 5). Bone healing was evaluated by qualitative and quantitative evaluation of serial radiographs every 2 weeks. Histopathological study was also performed on osteotomized radii upon completion of the experimental period. Dogs exposed to SMF had more advanced radiographic healing of osteotomy sites. Cellular morphology scores of the SMF group was significantly greater as compared with the control group (P<0.05). These results suggested that using the osteotomy gap model, SMF enhanced the radiographic and histopathological aspects of bone healing in dogs. Dogs at risk for delayed healing of fractures may benefit from treatment with SMF.

Evaluation of the Herb, Cissus quadrangularis in Accelerating the Healing Process of Femur Osteotomies in dogs

Maiti, S.K., Saravanan, B., Singh, G.R., Kumar, N., Hoque, M., Lal, J. and Kalicharan. 2007. Evaluation of the herb, Cissus quadrangularis in accelerating the healing process of femur osteotomies in dogs. J. Appl. Anim. Res, 31: 47–52. To evaluate the efficacy of the herb, Cissus quadrangularis Linn as an adjunct to internal fixation in bone healing in dogs, unilateral comminuted diaphyseal femoral osteotomy was created under general anaesthesia and immobilized with neutralization bone plate. All animals received routine postoperative treatment. In group B, additionally ethanolic extract of the herb was applied on skin surface twice a day along with subcutaneous injection (50 mg/kg body wt) on every alternate day for 20 days postoperatively. Early resolution of inflammatory signs and weight bearing were observed in animals of group B. Accelerated bone healing with complete bridging of comminuted fragments, extensive bony deposition along with periosteal and medullary continuity across the osteotomies site in the animals of group B were observed. Whereas, the process was relatively slow and the union was incomplete on day 60 in the animals of group A (control). It is concluded that Cissus quadrangularis accelerated the fracture healing.

Recombinant Human Bone Morphogenetic Protein‐2 in Absorbable Collagen Sponge Enhances Bone Healing of Tibial Osteotomies in Dogs

To compare the efficacy of 2 doses of recombinant human bone morphogenetic protein-2 (rhBMP-2) on tibial osteotomy healing in dogs. Experimental, randomized complete block (n=7). Adult female dogs (n=21). Right midshaft tibial osteotomies were created and stabilized with a 1-mm gap using type I external fixators. Seven dogs were untreated controls and 14 with osteotomies were treated with either 0.05 or 0.2 mg/mL rhBMP-2 delivered in an absorbable collagen sponge (ACS). At 8 weeks, dogs were euthanatized and bones were mechanically tested and examined by microscopy. Bone healing based on radiographic scoring, was significantly improved in dogs treated with 0.2 mg/mL of rhBMP-2 compared with the other groups; these tibiae were also significantly stronger and stiffer than 0.05 mg/mL rhBMP-2 and control osteotomized tibiae. Histologic scores were significantly better for 0.2 mg/mL rhBMP-2 group than 0.05 mg/mL rhBMP-2 group, but neither was significantly different from control. rhBMP-2 in ACS at a concentration of 0.2 mg/mL improves healing of tibial osteotomies in dogs compared with untreated controls and 0.05 mg/mL rhBMP-2 based on force plate analysis and radiographic evaluation. This was not confirmed histologically but treated bones had improved mechanical properties at 8 weeks. After a long bone fracture, dogs may face a long recovery period before full return of limb function. rhBMP-2, in association with good fracture fixation principles, may enhance bone healing in dogs with diaphyseal fractures.

Elastic cartilage grafting in canine radial fracture

Summary Bone has a capability to repair itself when it is fractured. Repair involves the generation of intermediate tissues, such as fibrous connective tissue, cartilage and woven bone, before final bone healing can occur. The process of cartilage-to-bone transition (CBT) is a key for the achievement of rigid bone healing during fracture repair. We tested this potential for elastic cartilage using a long bone defect model in dog. Eight sexually intact female mongrel dogs, 4.57 ± 0.53 years old and weighing 11.48 ± 0.63 kg, were studied. After an ostectomy of the midshaft radius, bone healing was evaluated over an 8-week period in control dogs (n = 4) and dogs in which autologous grafts of auricular cartilage were inserted into the bone defects (n = 4). Quantitative radiographic assessment was conducted every 2 weeks. Eight weeks post-operative, qualitative histopathologic analysis was performed on the operated radii. Furthermore, histological grading was done using the Ulutas et al., scoring system. Experiment dogs had more advanced radiographic healing of ostectomy sites. The defects with elastic cartilage implants were bridged completely with new bony spicules originated from the implants. Transformation of elastic cartilage clusters to mesenchymal connective tissue and bony spicules was obvious in the experiment group. Significant differences were observed for cellular morphology [3 ± 0.82 (experiment) vs. 1.75 ± 0.5 (control)] and cartilage integration [2 (experiment) vs. 1 (control)] at ostectomy sites between the studied groups. This study demonstrated that by using the ostectomy gap model, autologous auricular cartilage enhanced the radiographic and histopathologic aspects of bone healing in dogs.

Metabolic and histologic effects of recombinant canine somatotropin on bone healing in dogs, using an unstable ostectomy gap model

Abstract Objective To investigate the effect of recombinant canine somatotropin (STH) on the metabolic and histologic aspects of bone healing in dogs, using an unstable os-tectomy gap model. Animals 8 mature dogs. Procedure A 3-mm ostectomy of the mid portion of the radius was performed in all dogs. Implants designed to release STH at a rate of 4 mg/d were placed SC in 4 dogs (treated group [STHG]), and another 4 dogs received no implants (control group ICG]). Serum concentrations of STH, insulin-like growth factor I, and osteocalcin were de-termined before surgery, and weekly for 8 weeks. Scinti-graphic evaluation of the ostectomy sites was performed before surgery, and at weeks 2, 4, 6, and 8 after surgery. Histologic evaluation of the ostectomy sites was performed at the conclusion of the study at week 8. Results Significant (P &lt; 0.05) increases in serum STH, insulin-like growth factor I, and osteocalcin concentrations were observed in dogs of the STHG during the 8-week study period. Scintigraphic activity of the ostectomy sites was increased in dogs of both groups, but dogs of the STHG had significantly (P &lt; 0.05) greater activity, compared with dogs of the CG. Coalescence of nuclear activity across the ostectomy site was observed in dogs of the STHG, whereas dogs of the CG maintained 2 distinct areas of metabolic activity. Histologically, dogs of the STHG had bridging calluses with areas of endochondral ossification and ongoing osteogenic activity, whereas dogs of the CG had nonossified fibrocartilage typical of nonunion fractures. Conclusion Using the ostectomy gap model, recombinant canine STH enhanced the metabolic and histologic aspects of bone healing in dogs. (Am J Vet Res 1996;57:1395–1401)

Protein-Deficient Diet Alters Serum Alkaline Phosphatase, Bile Acids, Proteins and Urea Nitrogen in Dogs

Inadequate dietary protein intake in dogs can occur in a number of clinical situations, including prolonged voluntary anorexia, feeding of improperly formulated homemade diets (i.e., vegetarian) and overzealous protein restriction for the management of hepatic dis ease, renal insufficiency or struvite urolithiasis. Pre liminary data in our laboratory and others (Osborne et al. 1985) suggested that restricted protein diets (8% dry matter basis (DMB) ) can result in mild abnormal ities in a number of common serum biochemical assays including serum albumin, urea nitrogen and alkaline phosphatase. For that reason, a study was undertaken to assess the effect of an overtly protein-deficient diet on serum biochemical assays and hepatic function tests. Materials and methods. A prospective random ized study was designed to evaluate the effects of a protein-deficient diet on serum proteins, urea nitro gen, hepatic enzymes and bile acids. This experiment was one component of a larger study evaluating the effects of protein deficiency on bone healing in dogs. The experimental protocol was reviewed and approved by the institutional Animal Care and Use Committee of the Akron City Hospital. Sixteen healthy adult female beagles were utilized in the study. Dogs were determined to be healthy be fore the study on the basis of a physical examination, complete blood count, serum biochemistry panel and urinalysis. Eight of the dogs consumed a control diet for the entire 6-mo course of the study. The remaining eight dogs were fed a protein-deficient diet. Dogs were randomly assigned to each feeding group. Dogs were housed individually and were meal fed an amount cal culated to maintain their body weight. To detect ex ternal evidence of protein malnutrition, physical ex ams were performed on each dog weekly. Body weights were also recorded weekly. Table 1 outlines the nutrient profile and metabolizable energy content of the two experimental diets used in the study. Table 2 provides the ingredient composition of the two diets. Both diets were for mulated and manufactured by the Experimental Food Laboratory at Mark Morris Associates (Topeka, KS) using a twin screw extruder (APV Baker, Grand Rap ids, MI) for the cooking and forming process. The amino acid sources in both the control and pro tein-deficient diets were egg and food grade cornstarch. Egg was an important source of protein in both the protein-deficient (80%) and control (95%) diets. Amino acids contained in the cornstarch supplied ~20% and 5% of the protein in the protein-deficient and control diets, respectively. The amino acid profile of the control diet exceeded the canine amino acid re quirements established by Association of American Feed Control Officials (1994) and the National Re search Council (1985). Jugular venipuncture was performed after witholding food for 12 h at 0 (baseline), 4, 8, 12, 16 and 24 wk of the study. Venous blood was also sampled preand postprandially (2 h) during weeks 12 and 24 for serum bile acid analysis. Serum was harvested im mediately and frozen at -70°C pending analysis.