Exposure to Secondhand Smoke Impairs Fracture Healing in Rats.

BACKGROUND: Nonsmokers may be affected by environmental tobacco smoke (secondhand smoke), but the effects of such exposure on fracture healing have not been well studied. QUESTIONS/PURPOSES: To explore the possible effects of passive inhalation of tobacco smoke on the healing of a diaphyseal fracture in femurs of rats. We hypothesized that secondhand exposure to tobacco smoke adversely affects fracture healing. METHODS: A mid-diaphyseal fracture was created in the femur of 41 female Wistar rats and fixed with an intramedullary metallic pin; 14 rats were excluded (nine for inadequate fractures and five for K wire extrusion). Tobacco exposure was provided by a smoking machine on a daily basis of four cigarettes a day. Each cigarette yielded 10 mg tar and 0.8 mg nicotine, and was puffed by alternating injections of fresh air for 30 seconds and smoke air for 15 seconds. The smoke exposure was previously adjusted to provide serum levels of cotinine similar to human secondhand tobacco exposure. Cotinine is a predominant catabolite of nicotine that is used as a biological biomarker for exposure to tobacco smoke. In one group (n = 11), the animals were intermittently exposed to tobacco smoke before sustaining the fracture but not afterward. In another group (n = 7), the exposure occurred before and after the fracture. The control group (n = 9) was sham-exposed before and after the fracture. We evaluated the specimens 28 days after bone fracture. The callus quality was measured by dual-energy x-ray absorptiometry (bone mineral density [BMD], bone mineral content [BMC], and callus area), µCT (callus volume and woven bone fraction), and mechanical bending (maximum force and stiffness). RESULTS: Tobacco exposure resulted in delayed bone callus formation, which is represented by decreased BMD (Control: 0.302 ± 0.008 g/cm2 vs Preexposed: 0.199 ± 0.008 g/cm2 and Pre- and Postexposed: 0.146 ± 0.009 g/cm2; mean difference = 0.103 g/cm2, 95% CI, 0.094-0.112 g/cm2and mean difference = 0.156 g/cm2, 95% CI, 0.147-0.167 g/cm2; p < 0.01), BMC (Control: 0.133 ± 0.005 g vs Preexposed: 0.085 ± 0.0034 g and Pre- and Postexposed: 0.048 ± 0.003 g; mean difference = 0.048 g, 95% CI, 0.045-0.052 g and mean difference = 0.085 g, 95% CI, 0.088-0.090 g; p < 0.01), callus volume (Control: 7.656 ± 1.963 mm3 vs Preexposed: 17.952 ± 1.600 mm3 and Pre- and Postexposed: 40.410 ± 3.340 mm3; mean difference = -10.30 mm3, 95% CI, -14.12 to 6.471 mm3 and mean difference, -32.75 mm3, 95% CI, -36.58 to 28.93 mm3; p < 0.01), woven bone fraction (Control: 42.076 ± 3.877% vs Preexposed: 16.655 ± 3.021% and Pre- and Postexposed: 8.015 ± 1.565%, mean difference = 0.103%, 95% CI, 0.094-0.112% and mean difference = 0.156%, 95% CI, 0.147-0.166%; p < 0.01), maximum force (Control: 427.122 ± 63.952 N.mm vs Preexposed: 149.230 ± 67.189 N.mm and Pre- and Postexposed: 123.130 ± 38.206 N.mm, mean difference = 277.9 N.mm, 95% CI, 201.1-354.7 N.mm and mean difference = 304 N.mm, 95% CI, 213.2-394.8 N.mm; p < 0.01) and stiffness (Control: 491.397 ± 96.444 N.mm/mm vs Preexposed: 73.157 ± 36.511 N.mm/mm and Pre- and Postexposed: 154.049 ± 134.939 N.mm/mm, mean difference = 418.2 N.mm/mm, 95% CI, 306.3-530.1 N.mm/mm and mean difference = 337.3 N.mm/mm, 95% CI, 188.8-485.9 N.mm/mm; p < 0. 01). CONCLUSIONS: Rats exposed to tobacco smoke showed delayed fracture healing and callus that was characterized by decreased maturity, density, and mechanical resistance, which was confirmed by all assessment methods of this study. Such effects were more evident when animals were exposed to tobacco smoke before and after the fracture. Future studies should be done in human passive smokers to confirm or refute our findings on fracture callus formation. CLINICAL RELEVANCE: The potential hazardous effects of secondhand smoke on fracture healing in rodents should stimulate future clinical studies in human passive smokers.

Analysis of fracture healing in osteopenic bone caused by disuse: experimental study

Osteoporosis has become a serious global public health issue. Hence, osteoporotic fracture healing has been investigated in several previous studies because there is still controversy over the effect osteoporosis has on the healing process. The current study aimed to analyze two different periods of bone healing in normal and osteopenic rats. Sixty, 7-week-old female Wistar rats were randomly divided into four groups: unrestricted and immobilized for 2 weeks after osteotomy (OU2), suspended and immobilized for 2 weeks after osteotomy (OS2), unrestricted and immobilized for 6 weeks after osteotomy (OU6), and suspended and immobilized for 6 weeks after osteotomy (OS6). Osteotomy was performed in the middle third of the right tibia 21 days after tail suspension, when the osteopenic condition was already set. The fractured limb was then immobilized by orthosis. Tibias were collected 2 and 6 weeks after osteotomy, and were analyzed by bone densitometry, mechanical testing, and histomorphometry. Bone mineral density values from bony calluses were significantly lower in the 2-week post-osteotomy groups compared with the 6-week post-osteotomy groups (multivariate general linear model analysis, P<0.000). Similarly, the mechanical properties showed that animals had stronger bones 6 weeks after osteotomy compared with 2 weeks after osteotomy (multivariate general linear model analysis, P<0.000). Histomorphometry indicated gradual bone healing. Results showed that osteopenia did not influence the bone healing process, and that time was an independent determinant factor regardless of whether the fracture was osteopenic. This suggests that the body is able to compensate for the negative effects of suspension.

Analysis of fracture healing in osteopenic bone caused by disuse: experimental study.

Osteoporosis has become a serious global public health issue. Hence, osteoporotic fracture healing has been investigated in several previous studies because there is still controversy over the effect osteoporosis has on the healing process. The current study aimed to analyze two different periods of bone healing in normal and osteopenic rats. Sixty, 7-week-old female Wistar rats were randomly divided into four groups: unrestricted and immobilized for 2 weeks after osteotomy (OU2), suspended and immobilized for 2 weeks after osteotomy (OS2), unrestricted and immobilized for 6 weeks after osteotomy (OU6), and suspended and immobilized for 6 weeks after osteotomy (OS6). Osteotomy was performed in the middle third of the right tibia 21 days after tail suspension, when the osteopenic condition was already set. The fractured limb was then immobilized by orthosis. Tibias were collected 2 and 6 weeks after osteotomy, and were analyzed by bone densitometry, mechanical testing, and histomorphometry. Bone mineral density values from bony calluses were significantly lower in the 2-week post-osteotomy groups compared with the 6-week post-osteotomy groups (multivariate general linear model analysis, P<0.000). Similarly, the mechanical properties showed that animals had stronger bones 6 weeks after osteotomy compared with 2 weeks after osteotomy (multivariate general linear model analysis, P<0.000). Histomorphometry indicated gradual bone healing. Results showed that osteopenia did not influence the bone healing process, and that time was an independent determinant factor regardless of whether the fracture was osteopenic. This suggests that the body is able to compensate for the negative effects of suspension.

Diabetes mellitus affects the biomechanical function of the callus and the expression of TGF-beta1 and BMP2 in an early stage of fracture healing.

Transforming growth factor beta 1 (TGF-ß1) and bone morphogenetic protein-2 (BMP-2) are important regulators of bone repair and regeneration. In this study, we examined whether TGF-ß1 and BMP-2 expressions were delayed during bone healing in type 1 diabetes mellitus. Tibial fractures were created in 95 diabetic and 95 control adult male Wistar rats of 10 weeks of age. At 1, 2, 3, 4, and 5 weeks after fracture induction, five rats were sacrificed from each group. The expressions of TGF-ß1 and BMP2 in the fractured tibias were measured by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction, weekly for the first 5 weeks post-fracture. Mechanical parameters (bending rigidity, torsional rigidity, destruction torque) of the healing bones were also assessed at 3, 4, and 5 weeks post-fracture, after the rats were sacrificed. The bending rigidity, torsional rigidity and destruction torque of the two groups increased continuously during the healing process. The diabetes group had lower mean values for bending rigidity, torsional rigidity and destruction torque compared with the control group (P<0.05). TGF-ß1 and BMP-2 expression were significantly lower (P<0.05) in the control group than in the diabetes group at postoperative weeks 1, 2, and 3. Peak levels of TGF-ß1 and BMP-2 expression were delayed by 1 week in the diabetes group compared with the control group. Our results demonstrate that there was a delayed recovery in the biomechanical function of the fractured bones in diabetic rats. This delay may be associated with a delayed expression of the growth factors TGF-ß1 and BMP-2.

Diabetes mellitus affects the biomechanical function of the callus and the expression of TGF-beta1 and BMP2 in an early stage of fracture healing

Transforming growth factor beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) are important regulators of bone repair and regeneration. In this study, we examined whether TGF-β1 and BMP-2 expressions were delayed during bone healing in type 1 diabetes mellitus. Tibial fractures were created in 95 diabetic and 95 control adult male Wistar rats of 10 weeks of age. At 1, 2, 3, 4, and 5 weeks after fracture induction, five rats were sacrificed from each group. The expressions of TGF-β1 and BMP2 in the fractured tibias were measured by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction, weekly for the first 5 weeks post-fracture. Mechanical parameters (bending rigidity, torsional rigidity, destruction torque) of the healing bones were also assessed at 3, 4, and 5 weeks post-fracture, after the rats were sacrificed. The bending rigidity, torsional rigidity and destruction torque of the two groups increased continuously during the healing process. The diabetes group had lower mean values for bending rigidity, torsional rigidity and destruction torque compared with the control group (P<0.05). TGF-β1 and BMP-2 expression were significantly lower (P<0.05) in the control group than in the diabetes group at postoperative weeks 1, 2, and 3. Peak levels of TGF-β1 and BMP-2 expression were delayed by 1 week in the diabetes group compared with the control group. Our results demonstrate that there was a delayed recovery in the biomechanical function of the fractured bones in diabetic rats. This delay may be associated with a delayed expression of the growth factors TGF-β1 and BMP-2.

Mechanical properties of radial bone defects treated with autogenous graft covered with hydroxyapatite in rabbit.

PURPOSE: To determine biomechanical property of autogenous bone graft covered with hydroxyapatite in the defect of radial bone in rabbit. METHODS: Eighteen adult male New Zealand white rabbits were used which were divided into three groups (I, II, III) of six rabbits each. A segmental bone defect of 10 mm in length was created in the middle of the right radial shaft under general effective anesthesia in all rabbits and were stabilized using mini-plate with four screws. The defects In group I were left as such without filler, whereas in group II the defect were filled up with harvested 10 mm rib bone and in group III the defect were packed with rib bone covered with nano-hydroxyapatite. All rabbits in three groups were divided into two subgroups (one month and three months duration with three rabbits in each one). RESULTS: The mechanical property and the mean load for fracturing normal radial bone was recorded 388.2 ± 6 N whereas it was 72.4 ± 12.8 N for group I in 1 month duration which was recorded 182.4 ± 14.2 N for group II and 211.6 ± 10.4 N for group III at the end of 1 month. These values were 97.6 ± 10.2 N for group I and 324.6 ± 8.2 N for group II and 372.6 ± 17.4 N for group III at the end of three months after implantation. CONCLUSION: Implantation of autologous graft covered with hydroxyapatite indicated to have positive effect in integral formation of qualitative callus at the site of fracture and early re-organization of callus to regain mechanical strength too.

Mechanical properties of radial bone defects treated with autogenous graft covered with hydroxyapatite in rabbit / Propriedades mecânicas de defeito de osso radial tratado com enxerto autógeno coberto com nano-hydroxyapatite em coelho

PURPOSE: To determine biomechanical property of autogenous bone graft covered with hydroxyapatite in the defect of radial bone in rabbit. METHODS: Eighteen adult male New Zealand white rabbits were used which were divided into three groups (I, II, III) of six rabbits each. A segmental bone defect of 10 mm in length was created in the middle of the right radial shaft under general effective anesthesia in all rabbits and were stabilized using mini-plate with four screws. The defects In group I were left as such without filler, whereas in group II the defect were filled up with harvested 10 mm rib bone and in group III the defect were packed with rib bone covered with nano-hydroxyapatite. All rabbits in three groups were divided into two subgroups (one month and three months duration with three rabbits in each one). RESULTS: The mechanical property and the mean load for fracturing normal radial bone was recorded 388.2±6 N whereas it was 72.4±12.8 N for group I in 1 month duration which was recorded 182.4±14.2 N for group II and 211.6±10.4 N for group III at the end of 1 month. These values were 97.6±10.2 N for group I and 324.6±8.2 N for group II and 372.6±17.4 N for group III at the end of three months after implantation. CONCLUSION: Implantation of autologous graft covered with hydroxyapatite indicated to have positive effect in integral formation of qualitative callus at the site of fracture and early re-organization of callus to regain mechanical strength too.

OBJETIVO: Determinar as propriedades biomecânicas de enxerto ósseo autógeno coberto com hidroxiapatita em defeito do osso radial em coelhos. MÉTODOS: Foram utilizados 18 coelhos adultos, machos, brancos, Nova Zelândia, distribuídos em três grupos (I, II, III) de seis coelhos cada. Um defeito segmentar de 10 mm de comprimento foi criado no meio do eixo radial direito sob anestesia geral efetiva em todos os coelhos e foram fixados usando mini-placa com quatro parafusos. Os defeitos no grupo I foram deixados sem preenchimento, enquanto no grupo II o defeito foi preenchido com 10 mm de costela recoberta com nano-hidroxiapatita. Os coelhos nos três grupos foram distribuídos em dois subgrupos (Um e três meses, com três coelhos cada um). RESULTADOS: A propriedade mecânica e a média do peso para fraturar o osso radial normal foi 388,2±6 N, enquanto para o Grupo I, com um mês foi 72,4±12,8 N, para o grupo II 182,4±14,2 N e para o Grupo III 211,6±10,4 N. Após três meses de implantação, os valores foram 97,6±10,2 N para o Grupo I, 324,6±8,2 N para o Grupo II e 372,6±17,4 N para o Grupo III. CONCLUSÃO: A implantação de enxerto autólogo recoberto com hidroxiapatita indicou ter um efeito positivo na formação integral qualitativa do calo ósseo no local da fratura e precoce reorganização do calo com recuperação da força mecânica.

Healing of displaced condylar process fracture in rats submitted to protein undernutrition.

INTRODUCTION: This study evaluated the healing of mandibular condylar fracture in rats submitted to experimental and protein undernutrition (8% of protein) by means of histological analysis. MATERIAL: Forty-five adult Wistar rats were divided into three groups of 15 animals: a fracture group, who were submitted to condylar fracture with no changes in diet; an undernourished fracture group, who were submitted to a low protein diet and condylar fracture: an undernourished group, kept until the end of experiment, without condylar fracture. Displaced fractures of the right condyle were created under general anaesthesia. The histological study comprised fracture site and temporomandibular joint evaluations. RESULTS: The undernourished fracture group showed significant weight loss. There was a marked decrease in the values of serum proteins and albumin in the undernourished fracture group. Histological analysis showed that protein undernutrition lead to atrophy of the condylar fibrocartilage. Fractures in undernutrition presented a delay in callus formation due to more extensive devitalized bone areas, and after 3 months there were still bone formation areas, while fibrous ankylosis occurred in the articular space. CONCLUSION: It was concluded that mandibular condyle fractures in rats with protein undernutrition had impaired callus formation, as well as fibrous ankylosis into the temporomandibular joint.

Resposta osteogênica sistêmica no estímulo medular ósseo / Osteogenic systemic response at bone marrow stimulation

OBJETIVO: Avaliar a influência da resposta osteogênica sistêmica, causada pelo estímulo da medula óssea à distância, na consolidação de falha óssea. MÉTODO: 36 coelhos adultos jovens foram divididos aleatoriamente em três grupos (A, B, C) e submetidos à ostectomia do rádio direito, com retirada de 4mm de fragmento ósseo. Os animais do grupo A foram submetidos ao estímulo da medula óssea pela sua ablação do fêmur esquerdo. Os animais do grupo B foram submetidos ao estímulo da medula óssea pela introdução de fio de Kirschner com 1,5mm de espessura, no interior do canal medular femoral esquerdo. Os animais do grupo C foram utilizados como grupo controle. Foram realizadas radiografias semanais até a 4ª semana pós-operatória, quando os animais foram sacrificados. Foi realizado estudo histomorfométrico do calo ósseo formado no local da ostectomia. As radiografias foram avaliadas para análise da evolução da consolidação óssea. RESULTADOS: Os grupos que sofreram estímulo medular à distância tiveram menor número de células ósseas, comparativamente ao grupo controle. No estudo radiográfico não houve diferença na evolução da consolidação entre os grupos. CONCLUSÃO: O estímulo da medula óssea à distância influenciou desfavoravelmente a consolidação de falha óssea em coelhos.

OBJECTIVE: To assess the influence of systemic osteogenic response caused by remote stimulation of bone marrow in a bone gap union. METHOD: 36 young adult rabbits were employed. The animals were randomly divided into 3 groups (A, B, C) and submitted to ostectomy of the right radius, removing 4mm of bone. The animals on Group A had their bone marrow stimulated by ablation on the left femur. Animals on Group B had their bone marrow stimulated by introducing a 1.5mm-thick Kirschner wire into the shaft of the left femur. The animals on Group C served as controls. X-ray images were taken on a weekly basis until the 4th post-surgical week, when the animals were sacrificed. Histomorphometric study of the bony callus formed at the ostectomy site was conducted. The x-ray images were evaluated in order to analyze the evolution of bone union at the ostectomy site. RESULTS: The groups with remote bone marrow stimulation had a smaller number of bone cells as compared to the control group. On radiographic studies, no difference in terms of evolution of union was evident between the groups. CONCLUSION: Remote stimulation of bone marrow had an unfavorable influence on bone gap union in rabbits.

O uso do aspirado de medula óssea de ilíaco em falhas ósseas de fêmures de camundongos: estudo experimental / The use of inhaled bone marrow of ileum in bone failures of femurs of rats: experimental study

Os autores estudam a utilizacão de medula óssea em camundongos como estimulacão da formacão de calo ósseo. Foram utilizados dez camundongos adultos machos de linhagem isogênica gioto com peso de aproximadamente 250 gramas, e realizadas falhas ósseas na região distal do fêmur com alternância do lado direito e esquerdo, divididos em grupos A e B, sendo como controle camundongos com falha óssea isolado e com falhas ósseas com medula óssea colhida previamente de cada camundongo. Após análise qualitativa e quantitativa foi observado que o uso do aspirado de medula óssea não leva à estimulacão da formacão do calo ósseo e não há o aumento de processo inflamatório local.

Immediate placement of implants into periodontally infected sites in dogs. Part 2: A fluorescence microscopy study.

PURPOSE: Polychromatic sequence labeling of bone was used to study the effect of periodontal infection on the immediate placement of Frialit-2 implants. MATERIALS AND METHODS: In the surgical first phase, periodontitis was induced with ligatures involving the mandibular premolars of 5 mongrel dogs, and the contralateral teeth were used as controls (received only prophylaxis). After 3 months, the second phase was initiated and 40 implants were placed in the alveoli of both experimental and control teeth. During the healing period, fluorescent bone markers were injected to study bone formation around the implants. The dyes were injected in the following sequence: oxytetracycline hydrochloride at 3 days after implant placement, calcein green 4 weeks after implant placement, oxytetracycline 8 weeks after implant placement, and alizarin red S 3 days before sacrifice. Following a healing period of 12 weeks, the animals were euthanized and the hemimandibles were removed, dissected, fixed, and prepared for histomorphometic analysis of the percentage of each bone marker present. RESULTS: Fluorescence microscopy showed a similar sequence of bone remodeling (Mann-Whitney test) for both groups: experimental group, 9% bone formation at 3 days, 29% at 4 weeks, 21.6% at 8 weeks, and 52% at 12 weeks; control group, 14% at 3 days, 35.2% at 4 weeks, 32.3% at 8 weeks, and 45.8% at 12 weeks. DISCUSSION: Remodeling in both groups had similar characteristics in the degree of bone formation. CONCLUSIONS: It was concluded that periodontal disease does not affect bone remodeling around immediate implants. Although the healing in periodontally infected sites was slower initially, it reached the levels of the non-diseased sites after 12 weeks.

Osteogenesis imperfecta and hyperplastic callus formation in a family: a report of three cases and a review of the literature.

Osteogenesis imperfecta is one of the most common groups of inherited disorders of connective tissue. Hyperplastic callus formation in patients with osteogenesis imperfecta after fracture or surgery is a rare occurrence that has often been misdiagnosed as osteosarcoma. Previous series reported that hyperplastic callus formation is more often present in osteogenesis imperfecta male patients, with white sclerae, and a negative family history of the disorder. This is the first time that this complication has been presented in three female siblings, with a positive family history of osteogenesis imperfecta type IV. An association between osteogenesis imperfecta type IV and hyperplastic callus formation is unclear. This association might, however, be a separate, specific subtype of osteogenesis imperfecta, with an unknown inheritance pattern.

Nonunion using a canine model.

The investigation involved a search for a model of atrophic nonunion. Fifty-two mature, adult, mongrel dogs were used to study the repair after creating a 0.5-cm bone defect in the mid-diaphysis of the radius. In addition, a 2-cm wide strip of periosteum was circumferentially resected from each osteotomy extremity. No immobilization was used thereafter. The reparative process was assessed by X-rays, histology, vascular injection, and scintigraphy. The dogs we distributed into three groups according to the time of follow-up (1, 3, and 6 months). Two kinds of repair were recognized after 3 months and were well-established after 6 months: disturbed healing with much callus (54%) and disturbed healing with absent or scanty callus (46%). In the first instance, the periosteum had regenerated and produced the external callus. The bone ends were capped with fibrocartilage; the vascularization around the defect was increased and displayed a well-defined vascular picture. In the healing pattern with absent external callus (atrophic nonunion), the bone defect was enlarged and filled with fibrous tissue, but there was no deficient vascularization in and around the osteotomy. Radioactivity counting showed an increased uptake around the osteotomy site in both types of repair, which persisted over time but was higher in the 1-month group. It was concluded that the present model yields a consistent pattern of a disturbed reparative process that mimics human cases of atrophic or hypertrophic nonunion. The differences between the two kinds of repair seemed to be related to the periosteal capacity of regeneration.

Efeitos da desnervaçäo periférica sobre a formaçäo do calo ósseo: estudo experimental em ratos / Effects of the peripheral denervation on fracture healing: experimental study in rats

Foi feito estudo experimental em 24 ratos de raça Wistar, com 30 dias de vida. Foram produzidas fraturas fechadas na diáfise dos metatarsais de ambas as patas, ao mesmo tempo em que se procedeu à neurectomia ciática esquerda, ao nível de regiäo glútea. O processo de consolidaçäo das fraturas foi analisado através de estudo histológico nas 1ª, 2ª, 3ª e 6ª semanas após a produçäo da fratura, comparando-se o lado desnervado com o contralateral. A desnervaçäo ciática nao resultou em alteraçöes no processo de formaçäo do calo ósseo.

Electrically-induced osteogenesis in external fixation treatment.

This animal model experiment is performed to evaluate how bipolar direct current of 20 microamp. affects osteogenesis once external fixation treatment is applied in different forms: stabilization, distraction, and compression. In all cases a secondary healing, typical of external fixation treatment, was found; but a more mineralized callus and an increase in osteoprogenitor cell proliferation and differentiation were obtained through the use of electrical stimulation especially in the compression group.