The repair of segmental bone defects with porous bioglass: an experimental study in goat

This study was exclusively conducted to evaluate healing of surgically created defects on the radius of adult Black Bengal goat after implantation of porous bioglass blocks and compare the process kinetics with normal healing. Twelve Black Bengal goats were divided randomly into two groups: control and experimental group implanted with bioglass blocks. Unicortical bone defects in radius were generated in all animals under aseptic condition. Local inflammatory reaction and healing of wound, radiological investigations, histological studies, oxytetracycline leveling and angiographic studies were performed up to 90th day post-operatively and compared with normal healing. It has been found that extensive new bone formation originating from host bone towards the implant whereas in control, the process was active from both the ends; the defect site appeared as homogenous nonfluorescent area. Thus, porous bioglass promoted bone formation over the entire extension of the defect independent of size of block in comparison to control group.     

A technique for creating critical-size defects in the metatarsus of sheep for use in investigation of healing of long-bone defects

OBJECTIVE: To develop a technique for use in investigation of healing of long-bone defects by creation of a critical-size defect in the left metarsal III and IV bone (metatarsus) of sheep. ANIMALS: 18 healthy adult sheep. PROCEDURE: Sheep were allocated to 4 groups (3, 3, 5, and 7 sheep in groups 1 to 4, respectively). An ostectomy with various segmental length-to-diaphyseal diameter ratios (0.5, 1.0, 2.0, and 2.0 for groups 1 to 4, respectively) was performed on the left metatarsus of each sheep. The defect was left empty in sheep of groups 1, 2, and 3, whereas the defect was filled with a massive corticocancellous bone autograft in sheep of group 4. RESULTS: All sheep tolerated the surgical procedure well and were able to use the affected limb the day after surgery. Radiographic and histologic examinations conducted 16 weeks after surgery revealed nonunion in all sheep of groups 1, 2, and 3, whereas consistent bone healing with abundant bone formation was observed in all sheep of group 4. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these findings suggests that the sheep metatarsal model is a critical-size defect model with low morbidity. It should allow the assessment of new technologies for bone regeneration in conditions closely mimicking the clinical setting. IMPACT FOR HUMAN MEDICINE: Use of this technique in sheep should be of benefit for the preclinical study of osteoconductive, osteoinductive, or osteogenic biomaterials for use in humans.     

Induction of a barrier membrane to facilitate reconstruction of massive segmental diaphyseal bone defects: an ovine model

OBJECTIVES: To report an ovine model that can be used to evaluate the efficacy of bone substitutes for repair of segmental diaphyseal bone defects. STUDY DESIGN: Experimental study. ANIMALS: Eleven 2-year-old Pré-Alpes Sheep. METHODS: Mid-diaphyseal metatarsal bone defects (25 mm long) were stabilized by a dynamic compression plate over a polymethylmethacrylate (PMMA) cement spacer, and by external coaptation. The PMMA spacer was removed at 6 weeks by incising the encapsulating membrane. The defect remained unfilled (Group 1; n=5) or was filled with morselized autologous corticocancellous graft (Group 2; n=6), the membrane sutured closed, and external coaptation applied for 6 months, when healing was evaluated. RESULTS: Radiographic, computed tomographic, and histologic examinations at 6 months after the 2nd surgery revealed non-union in ungrafted defects whereas grafted defects showed bone healing. The induced membrane had blood vessels, CBFA1+ cells, and very few macrophages entrapped in a collagenous tissue positive for type I collagen. CONCLUSION: This ovine metatarsal defect model resulted in a critical-size defect (non-union) that healed when grafted. The PMMA-induced membrane constrained the graft, was well vascularized, and may have osteogenic properties. CLINICAL RELEVANCE: This model may be useful to evaluate new strategies in bone tissue engineering because the PMMA-induced membrane may help confine bone morphogenetic proteins, skeletal stem cells, or other agents to the defect cavity where they could be useful to enhance bone formation.     

Molecular analysis of defect healing in rat diaphyseal bone.

Spatial expression of messenger ribonucleic acid (mRNA) for osteoblastic marker in drill hole defect healing of adult male rats was analyzed by in situ hybridization. The defect was filled with hematoma 3 days after surgery, expressing Type I collagen mRNA. Hematoma was replaced with fibrous tissue on day 7, and then with new trabecular bone on day 10, originated from the intra-medullary space, respectively. mRNA for Type I collagen, parathyroid hormone 1 receptor (PTHIR), and alkaline phosphatase (ALP) were expressed in the same cell population of fibrous tissue adjacent to newly-formed trabecular bone, and in osteoblasts lining the newly-formed trabecular bone. Hematopoietic marrow with osteoclasts subsequently invaded the region, also from the intra-medullary space, replacing all the new trabecular bone by day 21, except for a thin sub-periosteal layer. mRNA for Type I collagen, PTH1R and ALP was expressed on the periosteal surface of thin layer. Although cartilage formation was not histologically visible, mRNA for Type II collagen was weakly detected in the majority of osteoblasts lining the newly-formed trabecular bone.     

Tibial segmental bone defect treated with bone plate and cage filled with either xenogeneic composite or autologous cortical bone graft. An experimental study in sheep.

Tibia segmental defect healing in sheep were clinically, radiographically and histologically evaluated. Twelve young sheep aged four to five months were divided into two groups, G1 and G2. A 3.5 cm long segmental defect was created in the right tibial diaphysis with maintenance of the periosteum. The bone defects in both groups were stabilized with a bone plate combined with a titanium cage. In G1 the cage was filled with pieces of autologous cortical bone graft. In G2 it was filled with a composite biomaterial which consisted of inorganic bovine bone, demineralized bovine bone, a pool of bovine bone morphogenetic proteins bound to absorbable ultra-thin powdered hydroxyapatiteand bone-derived denaturized collagen. Except for one G1 animal, all of them showed normal limb function 60 days after surgery. Radiographic examination showed initial formation of periosteal callus in both groups at osteo-tomy sites, over the plate or cage 15 days postoperatively. At 60 and 90 days callus remodeling occurred. Histological and morphometric analysis at 90 days after surgery showed that the quantity of implanted materials in G1 and G2 were similar, and the quantity of new bone formation was less (p = 0.0048) and more immature in G1 than G2, occupying 51 +/- 3.46% and 62 +/- 6.26% of the cage space, respectively. These results suggest that the composite biomaterial tested was a good alternative to autologous cortical bone graft in this experimental ovine tibial defect. However, additional evaluation is warranted prior to its clinical usage.     

Correlation of serum alkaline phosphatase activity with the healing process of long bone fractures in dogs

BACKGROUND: Bone healing is monitored mainly by physical and serial radiologic examinations of the fracture site. However, it is sometimes difficult to distinguish a delayed union from a nonunion, and advanced imaging techniques may not be available. Serum biochemical markers of bone formation, such as alkaline phosphatase (ALP) activity, may be clinically useful in evaluating the progress of healing. OBJECTIVE: The purpose of this study was to correlate serial values of serum ALP activity with the process of fracture healing in dogs and to assess its potential as a postsurgical prognostic indicator. METHODS: Changes in serum ALP activity were studied in 83 dogs with closed long bone diaphyseal fractures treated surgically. Physical and radiologic examinations of the fracture site and determination of serum ALP activity and calcium (Ca) and phosphate (P) concentrations were performed on admission (day 0); postoperatively on days 10, 20, and 30; and subsequently on a monthly basis until bone union was completed or signs of nonunion were evident. The dogs were allocated into 3 groups with respect to the fracture healing progress as documented by physical and serial radiologic examination. RESULTS: Group A dogs (n=35) developed a medium-sized callus that led to bone union within 2 months. Group B dogs (n=36) had a hypertrophic callus and delayed union, within 3-5 months. Group C dogs (n=12) had slow progress in fracture healing, with minimal callus formation during a 2-month period. Changes in mean serum ALP activity followed the same pattern in groups A and B, reaching a maximum level on day 10. Group A values returned to normal within 2 months, at which point bone union was complete, whereas group B values remained increased and returned to normal within 3-5 months, thus correlating with delayed union. In Group C, mean serum ALP activities showed no significant changes during the 2-month follow-up period, consistent with failure of bone union (nonunion). Serum P and Ca changes followed a proportional and inverse pattern to ALP changes, respectively. CONCLUSION: Serial determination of serum ALP activity during fracture healing could be an additional tool in predicting fractures at risk of developing a nonunion, helping the clinician to choose the appropriate intervention.     

Direct current stimulation of bone production in the horse: preliminary study with a "gap healing" model

The effect of a 20-microA direct-current implantable bone growth stimulator (BGS) on bone production with a "gap healing" model in the horse was evaluated. The right and left 4th metatarsal bones (Mt-4) were used in 7 adult horses to create the "gap healing" model. A 4-mm section of the Mt-4 bone was resected bilaterally in each horse. The BGS was surgically placed into the 7 left Mt-4 defects. The 7 right Mt-4 defects served as controls. Six horses survived the 16-week experimental period. Signs of pain, decreased range of limb motion, or lameness was not observed in any animal during the 16 weeks. None of the animals showed complete healing radiographically. Four stimulated sites showed less periosteal reaction and 2 showed greater reaction than the 6 controls. The greatest amount of periosteal reaction or bone resorption was seen around the screws and plates in both groups. Uptakes of 99mTc-MDP in counts/pixel for control sites and stimulated sites were 7.90 and 8.25 in the "gap defect" and 5.19 and 5.06 in the areas adjacent to the gap defect. The ratio of uptake between the gap defect and adjacent area was 1.5 and 1.58 respectively. Biocompatability of the BGS was excellent; however, 1 horse had a broken cathode wire 5 cm from the generator capsule at 6 weeks. All polyethylene cathode sheaths were fluid filled at 16 weeks. The average mineralization rates were 1.57 +/- 0.34, 1.71 +/- 0.28 mm/day and bone formation activity was 0.0182 +/- 0.171, and 0.0168 +/- 0.0149 mm2/day for control limbs and stimulated limbs, respectively. There was no significant difference between groups in any of the histomorphometric values measured. Direct current (20 microA) did not increase bone production in this experiment. Methods to objectively evaluate electrically induced osteogenesis and a "gap defect" model for BGS research on the horse are discussed. The results provide a basis for additional research on electrical stimulation of fractures in the horse and for dose-response studies.     

Arthroscopic Subchondral Bone Plate Microfracture Technique Augments Healing of Large Chondral Defects in the Radial Carpal Bone and Medial Femoral Condyle of Horses

OBJECTIVE: To evaluate the effect of arthroscopic subchondral bone microfracture on healing of large chondral defects in horses. STUDY DESIGN: Short- (4 months) and long-term (12 months) in vivo experimental chondral defect model. ANIMALS: 10 horses, aged 2 to 5 years. METHODS: Each horse had a 1 cm2 full-thickness chondral defect created in both radial carpal bones and both medial femoral condyles. One carpus and one femoral condyle of each horse had the subchondral bone plate under the defect perforated using an orthopedic awl. All horses were exercised, five horses were evaluated after 4 months and five horses after 12 months. Gross, histologic, and histomorphometric examination of defect sites and repair tissues was performed, as was collagen typing of the repair tissue. RESULTS: On gross observation a greater volume of repair tissue filled treated defects (74%) compared with control defects (45%). Histomorphometry confirmed more repair tissue filling treated defects, but no difference in the relative amounts of different tissue types was observed. There was an increased percentage of type II collagen in treated defects compared with control defects and evidence of earlier bone remodeling as documented by changes in porosity. CONCLUSIONS: In full-thickness chondral defects in exercised horses, treatment with subchondral bone microfracture increased the tissue volume in the defects and the percentage of type II collagen in the tissue filling the defects when compared to nontreated defects. CLINICAL RELEVANCE: No negative effects of the microfracture technique were observed and some of the beneficial effects are the basis for recommending its use in patients cases with exposed subchondral bone.     

The effect of short-duration, high-intensity electromagnetic pulses on fresh ulnar fractures in rats

Pulsed electromagnetic fields (PEMFs) have been found to be beneficial to a wide variety of biological phenomena. In particular, PEMFs have been shown to be useful in the promotion of healing of ununited fractures. Conflicting information exists regarding the benefit of using PEMFs to accelerate the healing of fresh fractures. This paper reports on the evaluation of the effect of a new PEMF generator (PAP IMI) on the healing of fresh ulnar fractures in rats. This device is unique by virtue of the extremely high power output of each of the pulses it generates. Ulnar fractures were created in rats by using a bone cutter, thus producing a 2-3 mm bone defect. Rats were then randomly divided into treatment and control groups. The treatment group underwent periodic treatments with the PAP IMI, and the control group received no treatment. Radiographs of rats from both groups were taken at 1-week intervals. Histological evaluation was performed at the end of the study. Radiographic and histopathological evaluations were scored, and scores were used to assess both rate and quality of healing. The radiographic results demonstrated gradual bridging callus formation in both control and treatment groups, however, the healing process was faster in rats that were not treated by PEMF. Histological evaluation demonstrated that the fibrous content of the callus in rats belonging to the treatment group was significantly higher than that in rats belonging to the control group. The results of this study do not support the claim that PEMF generated by the PAP-IMI stimulate osteogenesis and bone healing after the creation of fresh ulnar fractures in rats.     

An animal model for interface tissue formation in cemented hip replacements

OBJECTIVE: To create a model in sheep for investigation of early changes related to the formation of an interface membrane in hip prosthesis. STUDY DESIGN: Experimental study. ANIMALS: Twenty-four female adult Swiss Alpine sheep. METHODS: Sheep were divided into 2 groups of 12 for unilateral cemented total hip arthroplasty. In Group I, the prosthesis was fixed with retrograde cement gun injection to achieve a complete cement mantle, whereas in Group II a primary cement mantle defect was produced. Groups I and II were further divided into 2 sub-groups with study end points of 2 and 8.5 months after surgery. Radiographs were evaluated postoperatively and at euthanasia for migration of the femoral component and bone resorption. Histologic sections were evaluated semiquantitatively for changes in cell types and numbers, and bone reactions; and quantitatively for size of interface membrane and new bone formation. RESULTS: Radiographically, there tended to be an increase in bone resorption and periosteal bone formation throughout the femoral shaft in Group II compared with Group I, but this was only statistically significant at the region of the femoral neck (R5) at both time periods (P<.05). Semiquantitative histologic evaluation revealed significant increases (P<.05) in cellularity, numbers of fibroblasts, giant cells, macrophages, and mononuclear cells, in Group II primarily at 2 months after surgery. This was also true for interface membrane formation and bone remodeling. Quantitative data showed an increased in the size of the interface membrane and area of bone formation at 8.5 months in Group II. CONCLUSIONS: The cement defect model offered controlled and repeatable production of an interface membrane. The results suggest that a primary cement mantle defect could be a possible trigger for implant instability, eliciting a cascade of biomechanical and molecular events in bone tissue leading to aseptic loosening. CLINICAL RELEVANCE: The results show the effect of defects in the cement mantle in promoting interface membrane formation. Long-term and biochemical studies are required to evaluate the relevance of this interface membrane formation.     

The Effects of Plaster of Paris and Autogenous Cancellous Bone on the Healing of Cortical Defects in the Femurs of Dogs

Four one quarter inch evenly spaced circular defects were created bilaterally in the lateral femoral diaphysis of 12 clinically normal adult dogs. The defects were left unfilled (control), or were filled with one of the following: (1) plaster of Paris, (2) an equal-volume mixture of plaster of Paris and autogenous cancellous bone, and (3) autogenous cancellous bone. The degree of bone healing was evaluated radiographically and histologically at 2, 4, 6, 8, 10, and 12 weeks. Radiographically, no objective conclusions could be drawn due to the small size of the defects and limited amount of plaster of Paris implanted. Histologically, there was no inflammatory reaction to the plaster of Paris. No differences were determined in the degree of bone healing between autogenous cancellous bone, plaster of Paris, and a mixture of plaster of Paris and autogenous cancellous bone. All implants were superior to the control defect in degree of bone healing.     

Comparable bone healing capacity of different bone graft matrices in a rabbit segmental defect model

We compared the bone healing capacity of three different demineralized bone matrix (DBM) products applied using different carrier molecules (hyaluronic acid [HA] vs. carboxymethylcellulose [CMC]) or bone compositions (cortical bone vs. cortical bone and cancellous bone) in a rabbit segmental defect model. Overall, 15-mm segmental defects in the left and right radiuses were created in 36 New Zealand White rabbits and filled with HA-based demineralized cortical bone matrix (DBX), CMC-based demineralized cortical bone matrix (DB) or CMC-based demineralized cortical bone with cancellous bone (NDDB), and the wound area was evaluated at 4, 8, and 12 weeks post-implantation. DBX showed significantly lower radiopacity, bone volume fraction, and bone mineral density than DB and NDDB before implantation. However, bone healing score, bone volume fraction, bone mineral density, and residual bone area at 4, 8, and 12 weeks post-implantation revealed no significant differences in bone healing capacity. Overall, three DBM products with different carrier molecules or bone compositions showed similar bone healing capacity.     

Effect of beta-tricalcium phosphate in surgically created subchondral bone defects in male horses

beta-Tricalcium phosphate (TCP) was evaluated as a synthetic bone grafting material in horses. Sixteen horses were randomly assigned to 4 groups of 4 horses. In each horse, an 11-mm by 10-mm circular defect with a smooth margin was created surgically in the weight-bearing surface of both third metacarpal bones (McIII) and the left third metatarsal bone. The defect in the left McIII was filled with TCP, the defect in the right McIII was filled with a mixture of 50% autogenous cancellous bone (ACB) and 50% TCP, and the defect in the left third metatarsal bone was not filled to serve as a control. Nuclear imaging and radiographic evaluation of surgical sites were performed at postsurgical week (PSW) 4 and 13 in group-I horses and at PSW 26, 39, and 52 in groups-II, -III, and -IV horses, respectively. Horses in groups I, II, III, and IV were euthanatized at PSW 13, 26, 39, and 52, respectively, and specimens were harvested for histologic evaluation. Results were compared among treatment methods, as well as among time intervals. The greatest activity at defect sites was seen in group-I horses at PSW 4. Activity counts in limbs of group-I horses at PSW 13 and counts in limbs of group-II horses at PSW 26 were not significantly (P less than or equal to 0.05) different. Activity counts in groups-III and -IV horses at PSW 39 and 52, respectively, were less than those in groups I and II. However, there was no significant difference in activity counts between limbs implanted with TCP or ACB:TCP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of healing of the osteotomy gap after tibial tuberosity advancement with and without use of an autogenous cancellous bone graft

Objective: To evaluate and compare healing, with and without the use of bone graft, of the gap created during tibial tuberosity advancement (TTA). Study Design: Prospective study and case series. Animals: Dogs treated with TTA (n=67). Methods: Prospective study: Mediolateral radiographic projections (6 weeks and 4 months) after TTA without use of bone graft (group I, n=14) were compared with radiographs of consecutive TTA in which the gap was filled with autologous cancellous bone graft (group II, n=14). Two scoring techniques (A, B) were used. Score A was used to grade the overall osteotomy healing (0=no healing, 4=healed osteotomy). Score B evaluated, independently of each other, healing in 3 sites: proximal to the cage (B1), between cage and plate (B2), and distal to the plate (B3). Case series: nongrafted TTA (4–25 weeks, n=39) were evaluated for healing (Score A). Data was analyzed using t‐tests and ANOVA. Significance was set at P≤.05. Results: Prospective study: Score A, B2, and B3 showed no difference in healing between groups at 6.8 weeks and 4.2 months. Score B1 revealed, in both rechecks, a significantly higher density in group II. Case series: Radiographs at 11.59±5.99 weeks scored 3.3 (2–4). No healing related complications were observed. Conclusion: The osteotomy gap created during TTA healed within expected time regardless of bone graft use.     

Treatment of nonunions with nonglycosylated recombinant human bone morphogenetic protein-2 delivered from a fibrin matrix

OBJECTIVE: To report the results of the treatment of nonunions with nonglycosylated recombinant human bone morphogenetic protein-2 (nglBMP-2) delivered from a designed fibrin matrix. STUDY DESIGN: Experimental trial in rodents and prospective clinical study in dogs and cats with nonunion fractures. ANIMALS: Twenty adult female, albino, Sprague-Dawley rats; 8 client-owned cats and dogs. METHODS: After development of a fibrin matrix and evaluation of nglBMP-2 in a rodent femoral defect model, 8 consecutive long bone nonunion fractures (no progression in healing in > or = 3 months), were treated using 300 microg nglBMP-2 in a liquid fibrin precursor, injected into the defect gap after fracture revision and stabilization, or through a stab incision into the fracture site. The fibrin matrix was designed to clot in the wound after 60 seconds and to release the nglBMP-2 continuously over several days. RESULTS: Using only fibrin gel, 7% of the rat femoral defect was filled with new formed bone compared with 79% defect filling using 2 microg nglBMP-2 (P=.006). Five and 10 microg nglBMP in fibrin resulted in union of all femoral defects with complete filling of the gap with new bone. Bony bridging and clinical healing was achieved in 7 patients within 24 weeks of administration of nglBMP-2. CONCLUSIONS: Application of nglBMP-2 in a functional matrix can induce bone healing. Controlled release of nglBMP-2 from a fibrin matrix mimics the natural fracture hematoma. CLINICAL RELEVANCE: nglBMP-2/fibrin can successfully replace a cancellous bone autograft in fracture treatment with an associated reduction in graft donor site morbidity and surgical time.     

The effect of compacted cancellous bone grafting on the healing of subchondral bone defects of the medial femoral condyle in horses

Objective— To compare the quality of second-intention healing and that of compacting sternally harvested cancellous bone into subchondral bone defects of the medial femoral condyle in horses.
Study Design— A controlled experiment using a surgical technique that minimizes soft tissue trauma, customized for consistency among horses.
Animals or Sample Population— Ten horses, aged 2 to 5 years, free of hindlimb lameness and with radiographically normal stifles.
Methods— After a 12.7-mm-diameter × 19-mm-deep defect was created into randomly selected medial femoral condyles, bone and cartilage healing was evaluated over a 6-month period in control horses (  n = 5  ) and horses receiving a compacted cancellous bone graft (  n = 5  ). Healing was evaluated using lameness assessment, radiographic and microradiographic interpretation, arthroscopic appearance, percent bone fill, proteoglycan content, and histology.
Results— Six months after surgery, there was no significant difference between grafted and ungrafted defects with respect to lameness, radiographic score, or percent bone fill. Histologically, grafted defects were characterized by the presence of dead graft and secondary cyst formation in four defects. Ungrafted defects filled with fibrous tissue and no cyst formation were identified.
Conclusions— Grafted defects do not heal better than ungrafted defects, and lameness was not affected by surgical technique.
Clinical Significance— Cartilage healing is similar in grafted and ungrafted defects in the equine medial femoral condyle at 6 months, suggesting that surgical debridement alone of cystic structures remains the treatment of choice.     

Effect of prostaglandin E1 on the periosteal regional acceleratory phenomenon in fractured ribs: histomorphometric study in beagles

Transverse fractures were made surgically in the midshaft of the left 9th and 10th ribs in adult Beagles. A buffer vehicle (n = 4) or 0.2 mg of prostaglandin (PG) E1/day (n = 6) was injected into the fracture sites twice a day for 10 days, and dogs were euthanatized on day 30. Double-pulsed fluorescent labels were given with each of 2 fluorochrome markers--calcein before surgical treatment and oxytetracycline HCl before euthanasia. Histomorphometric analysis was carried out on specimens collected in adjacent regions of the healing defects. The surface extent and width of the osteoid on fractured (P less than 0.01, P less than 0.05, respectively) and nonfractured (P less than 0.05) sites in the treated group were greater than those in the nontreated group. The net loss of mineralizing surfaces was noticed on both ribs of both groups. Of 11 samples on the fractured side in the treated group, 4 contained periosteal new bone proliferation. There was increased osteoid formation and decreased mineralizing surfaces in the PGE1-treated group. Seemingly, administration of PGE1 induced bone matrix formation on periosteal envelope adjacent to a fracture site and its contralateral matching site.     

Implantation of canine umbilical cord blood-derived mesenchymal stem cells mixed with beta-tricalcium phosphate enhances osteogenesis in bone defect model dogs

This study was performed to evaluate the osteogenic effect of allogenic canine umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) mixed with beta-tricalcium phosphate (β-TCP) in orthotopic implantation. Seven hundred milligrams of β-TCP mixed with 1 × 10⁶ UCB-MSCs diluted with 0.5 ml of saline (group CM) and mixed with the same volume of saline as control (group C) were implanted into a 1.5 cm diaphyseal defect and wrapped with PLGC membrane in the radius of Beagle dogs. Radiographs of the antebrachium were made after surgery. The implants were harvested 12 weeks after implantation and specimens were stained with H&E, toluidine blue and Villanueva-Goldner stains for histological examination and histomorphometric analysis of new bone formation. Additionally, UCB-MSCs were applied to a dog with non-union fracture. Radiographically, continuity between implant and host bone was evident at only one of six interfaces in group C by 12 weeks, but in three of six interfaces in group CM. Radiolucency was found only near the bone end in group C at 12 weeks after implantation, but in the entire graft in group CM. Histologically, bone formation was observed around β-TCP in longitudinal sections of implant in both groups. Histomorphometric analysis revealed significantly increased new bone formation in group CM at 12 weeks after implantation (p < 0.05). When applied to the non-union fracture, fracture healing was identified by 6 weeks after injection of UCB-MSCs. The present study indicates that a mixture of UCB-MSCs and β-TCP is a promising osteogenic material for repairing bone defects.     

Legg—Perthes'' Disease in the Dog

Abstract— —Twenty seven dogs (two of them obtained by inbreeding) in which Legg-Perthes' disease was verified clinically and roentgenologically, were studied to elucidate the early changes encountered. Histological-histochemical methods, oxytetracycline (OTC) bone labelling and auto- and micro-radiography techniques were used. All of the twelve young dogs with unfused growth plates showed histologically and histochemically, especially in the proximal part of the epiphysial cartilage, areas filled with a structureless, metachromatically staining mass, cyst-like formations and derangement of the architecture as the initial changes of the disease. By using S³⁵ it was possible to confirm the histochemical findings that substances belonging to the mucopolysaccharide group were formed in the degenerated areas of the growth plate. Histologically, degenerative changes were demonstrable earlier in the metaphysis than in the epiphysis. Absence of OTC uptake preceded bone necrosis histologically. Oxytetracycline bone labelling showed that regeneration derived chiefly from the metaphysis as the epiphysial cartilage began to disappear. Using microradiography, it was possible to illustrate the bone structure, especially to reveal the flattening of the epiphysis and the broadening of the metaphysis. In the later phase of the disease extensive articular cartilage changes were recorded. Together with the other degenerative changes described, they contributed to the formation of secondary osteoarthritis.     

Guided bone regeneration with beta-tricalcium phosphate and poly L-lactide-co-glycolide-co-epsilon-caprolactone membrane in partial defects of canine humerus

This study was performed to evaluate the effect of beta-tricalcium phosphate and poly L-lactide-co-glycolide-co-epsilon-caprolactone (TCP/PLGC) membrane in the repair of partial bone defects in canine proximal humerus. Three adult mixed-breed dogs were used during the experimental period. The length of the defect was quarter of the full length of humerus, and width of the defect was quarter of middle diameter of the lateral aspect of humerus. The humeri of each dog were divided into treatment (TCP/PLGC) and control groups. The defect was covered with TCP/PLGC membrane in treatment group. To evaluate regeneration of the bone, computerized tomography (CT) and histopathologic examination were performed. The radiopaque lines were appeared at the original defect sites in TCP/PLGC group but below the original site in control at 4th week. Radiopacity and thickness of the defect sites, and radiopaque lines were more increased at 8th week than those of 4th week. Histopathologic findings revealed fibrous connective tissue migration into the defect and the migration inhibited the structure of new cortex to be placed in the original level in control whereas new cortex growth was found in the level of original line in TCP/PLGC group. However, the new cortical bone in the TCP/PLGC group was thinner and less organized than the adjacent intact cortex, and the amount of new cancellous bones were also scanty. The result suggested that TCP/PLGC membrane is a good guided bone regeneration material to restore the original morphology of humerus in partial defect.