Medullary bone matrix formation, mineralization, and remodeling related to the daily egg-laying cycle of Japanese quail: a histological and radiological study

A model is presented concerning the remodeling of medullary bone during the egg-laying cycle of hens. This model is partly based on earlier results that showed that the active period of medullary bone resorption that produces part of the calcium needed for the eggshell coincides with the period of medullary bone matrix formation. It was hypothesized that mineralization of this matrix takes place during the subsequent inactive period. The present study examines this model. The medullary bone matrix volume, regardless of the degree of mineralization, is quantified in quail hens at four time points during the egg-laying cycle. No significant changes in the medullary bone volume (i.e., matrix and bone) during the egg-laying cycle were found, implying that matrix formation and bone resorption are kept in balance within a time interval of only a few hours. In the radiological part of this study the whole body retention (WBR) of 99mTc(Sn)MDP is used as a measure of the amount of low calcium medullary bone matrix formed during the egg-laying cycle. This use of 99mTc(Sn)MDP is justified by an autoradiographic experiment that showed that 99mTc(Sn)MDP actually labels newly formed medullary bone. Localization of the radioactive label showed a high positive correlation with the localization of fluorescent tetracycline labels in the medullary bone. From 4 to 22 hours after ovulation, the amount of medullary bone matrix increases linearly in quail hens.(ABSTRACT TRUNCATED AT 250 WORDS)     

A radiographical and biomechanical study of demineralized bone matrix implanted into a bone defect of rat femurs with and without bone marrow

Repair of large bone defects represents a challenge to orthopedic surgery since autogenous graft is not available in large amounts. Demineralized bone matrix (DBM) which contains bone morphogenic protein, a potent osteoinductive glycoprotein, and collagen, an osteoconductive matrix, may be an effective substitute for these graft materials. Bone marrow which contains osteoprogenitor cells could potentiate the osteoinductive and osteoconductive properties of demineralized bone matrix. This study tested the ability of demineralized bone matrix with and without bone marrow to bridge large segmental defects, and evaluated the results both radiographically and biomechanically as compared to autogenous (isogeneic) cancellous bone graft. Demineralized bone-matrix segments implanted into a plated femoral segmental defect in rats resulted in firm union in most animals. Bone marrow significantly enhanced bone formation of demineralized bone-matrix implants at an early stage but with time, differences between bone marrow-augmented and bone marrow-deprived demineralized bone implants were no longer demonstrable radiographically and biomechanically. Newly formed bone had about 50% of the strength of the contralateral control bones. Femurs implanted with cancellous bone isografts had similar evidence of absolute union rate, radiographic and mechanical properties as DBM-implanted femurs.     

Structural and histomorphometric studies of iliac crest trabecular and cortical bone in autosomal dominant osteopetrosis: a study of two radiological types

Cylindrical iliac crest biopsies were obtained from 16 patients with autosomal dominant osteopetrosis after intravital double labeling with tetracycline, and compared with normal age- and sex-matched controls. Ten patients had the radiological type I (5 women, 5 men, aged 17-62 years, mean 42) characterized by diffuse, symmetrical osteosclerosis and enlarged thickness of the cranial vault. Six patients had type II (2 women, 4 men, aged 22-44 years, mean 36), where "Rugger Jersey Spine" and endobone are characteristic findings. Structural studies of cortical and trabecular bone were performed, and trabecular bone resorption and formation rates were studied using dynamic histomorphometry. The total biopsy length (C. Wi) were increased in type I (p less than 0.05), and unchanged in type II. Both types showed increased cortical width (Ct. Wi) (p less than 0.01 and p less than 0.05, respectively), and decreased fractional width of cancellous bone (Cn.Wi/C.Wi) (p less than 0.01 and p less than 0.05). The fractional trabecular bone volume (BV/TV) and trabecular thickness (Tb. Th) were both significantly increased in type I (p less than 0.05), while resorptive and formative indices of trabecular bone remodeling were normal. No difference was found in trabecular bone balance, which was slightly positive in both patients and controls. In type II osteopetrosis the eroded surfaces (OS/BS) were significantly increased (p less than 0.01), as was the total resorptive period RP) (p less than 0.05). The resorption depth (R.D.) was normal, while the resorption rate (MRR) was insignificantly decreased. Many big multinucleated osteoclasts were seen in this type suggesting defective resorptive function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in extracellular matrix vesicles during healing of rat tibial bone: a morphometric and biochemical study

Primary mineral formation in woven bone has been associated with the production of extracellular matrix vesicles. Previous studies have demonstrated an increase in phospholipid: Ca:Pi complexes (CPLX) immediately prior to hydroxyapatite formation. Since matrix vesicles are enriched in phosphatidylserine and PS is the major phospholipid in CPLX, the present study examined whether the morphologic appearance of matrix vesicles and initial formation of crystals within them could be correlated to changes in their phospholipid composition and metabolism. Ablation of the tibial marrow in rats was used as the model since this procedure induces endosteal repair with primary mineralization. The morphologic appearance of the matrix vesicles was assessed by morphometric analysis at the electron microscopic level. Matrix vesicles were divided into 4 categories: empty, amorphic, crystal, and rupture. There was time dependent decrease in the number of empty and amorphic matrix vesicles which correlated with an increase in crystal and rupture type. Distance from the calcification front decreased as more rupture-type vesicles were noted. In a parallel set of experiments, matrix vesicle-enriched membranes (MVEM) were isolated from homogenates of endosteal tissue removed from the treated tibia as well as from the contralateral control. There was an increase at 6 days in MVEM alkaline phosphatase and phospholipase A2 specific activities in both limbs, the magnitude of response being significantly greater in the treated legs. The phospholipid composition of the MVEM changed with time. SPH was highest at day 3, PS was detectable only in day 6 and 14 samples, and PC exhibited a time dependent decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Xenogenic demineralized bone matrix and fresh autogenous cortical bone effects on experimental bone healing: radiological, histopathological and biomechanical evaluation

Background  Bone grafting is used to enhance healing in osteotomies, arthrodesis, and multifragmentary fractures and to replace bony loss resulting from neoplasia or cysts. They are source of osteoprogenitor cells and induce bone formation and provide mechanical support for vascular and bone ingrowth. Autografts are used commonly but quantity of harvested bone is limited. The aim of this study is to evaluate autograft and new xenogenic bovine demineralized bone matrix (DBM) effects on bone healing process. Materials and methods  Twenty male White New Zealand rabbits were used in this study. In group I (n = 10) the defect was filled by xenogenic DBM and in autograft group the defect was filled by fresh autogenous cortical graft and fixed by cercelage wire. Radiological, histopathological and biomechanical evaluations were performed blindly and results scored and analyzed statistically. Results  Statistical tests did not reveal any significant differences between two groups on the 14th postoperative day radiographically (P > 0.05). There was a significant difference for union on 28th and 42nd postoperative days and for remodeling at on the 56th postoperative day radiologically (P < 0.05). Statistical tests did not support any significant differences between two groups for radiological bone formation (P > 0.05). Histopathological and biomechanical evaluation revealed no significant differences between two groups. Conclusions  The results of this study indicate that satisfactory healing occurred in rabbit radius defect filled with xenogenic bovine DBM. Complications were not identified and healing was faster, same as in cortical autogenous grafting.     

Bone healing in rabbits after compression osteosynthesis: a comparative study between the radiological and histological findings

The relationship between the radiological and histological findings was studied in a series of rabbit osteotomies and fractures stabilized with plates with and without longitudinal compression.

When compression was employed, the gap varied between 10 and 200 μm and no radiologically visible callus was seen. After 4 weeks the smallest gaps of 10–30 μm were filled histologically with structureless material, while the larger gaps contained ‘cortical’ bone derived from periosteal and endosteal proliferation. The degree of union could not be assessed from X-ray examination, as there was no correlation between the histological and radiological appearances.

When no compression was employed, the gap varied between 200–1200 μm; radiologically visible periosteal callus and filling of the gap indicated that consolidation had appeared in four weeks. Gaps of 200–500 μm were filled with bone derived from the periosteum and endosteum of lamellar structure which had invaded the original cortical bone (‘plugging’). When the gaps were more than 500 μm, islands of cartilage and connective tissue were found. In all cases studied, many ‘anchoring osteons’ passed from the endosteal and periosteal callus into the original cortical bone.

It is concluded that a ‘compression internal fixation’ of cortical bone will not necessarily secure union, as histologically confirmed, even when the formation of radiologically visible callus is suppressed. The lack of periosteal callus appears to be the result of a small gap and rigid fixation, rather than related to strains induced in the bone.     

Effect of a five-week swimming program on rat bone: A histomorphometric study

Summary To specify the exercise-induced changes on different skeletal sites, the effect of a 5-week endurance swin training was studied in rats. Eighteen Lyon strain (Sprague-Dawley) 5-week old female rats were divided into nine sedentary and nine swimming rats. Each swim training session was increased by 15 minutes from 2–6 hours per day. A histomorphometric study was performed at the primary and secondary spongiosa of the distal femur and at the secondary spongiosa of lumbar and thoracic vertebral bodies. After training, bone loss was observed in the secondary spongiosa of lumbar vertebral bodies (24.7%) and in the primary spongiosa of distal femur (15.2%). A tendency to bone loss was also detected in the secondary spongiosa of distal femur (10.8%), whereas no change was detected in thoracic vertebral bodies. In secondary spongiosa, bone loss was accompanied with a thinning of trabeculae. Total eroded surfaces and osteoid surfaces were significantly decreased in the three studied skeletal sites, suggesting a decreased bone turnover. The decreased thickness of osteoid seams in both lumbar vertebrae and distal femur could mean that the osteoblastic activity has also been altered at the cell level, leading to thinning of trabeculae. Five-week swim training with such duration and intensity of exercise appears unable to increase bone volume in rats and, therefore, causes adverse effects. The three studied bones seemed to adapt differently to experimental conditions. The lack of ground reaction forces induced by water immersion might have contributed to the observed bone loss. Normal gravity would be an important cofactor in the osteogenic effects of exercise.     

Effects of graded reaming on fracture healing: Blood flow and healing studied in rat femurs

In 30 rats, closed bilateral fractures of the femur were produced. On the left side intramedullary reaming was performed to 1.6 mm, and the fracture fixed with a steel pin with a diameter of 1.6 mm. On the right side the femoral canal was reamed to 2.0 mm and a hollow steel tube with a diameter of 2.0 mm was used for fixation. An additional 8 rats were used to obtain mechanical, dimensional and flow data on intact femurs, and another 10 rats were used to study the acute flow changes caused by fracture and different degrees of reaming and fracture.

Fracture and reaming reduced total bone and cortical bone blood flows to about one third of normal flow, with no differences between the 1.6-mm and the 2.0-mm reamed bones. At 4 weeks, total bone flow was about double and cortical bone flow about 4 times increased in the 1.6-mm group. In the 2.0-mm reamed bones increases of approximately 5 times in total bone flow and of about 7 times in cortical flow were found. Callus flow was about twice the size of the respective cortical flow in both groups. Both total and cortical flows gradually subsided, without differences between the 2 groups. At 12 weeks, the callus area in the 2.0-mm group was greater than in the 1.6-mm group, while bone dimensions were greater in the 2.0-mm group at 4 and 12 weeks. Bending moment and rigidity were greater in the 1.6-mm group than in the 2.0-mm one at every time interval; no differences were found in fracture energy.

We conclude that, in terms of healing, modest reaming is preferable to extensive reaming. The adverse effect of extensive reaming is not due to excessive flow derangement at the acute stage or to impaired vascularity at the phase of remodeling.     

Electromagnetic stimulation of bone repair: a histomorphometric study

The effect of pulsing electromagnetic fields (PEMFs) on bone repair was studied in principal metacarpal bones of eight adult male horses: Six horses were treated with PEMFs, and two horses were untreated. In treated horses, Helmholtz coils were applied during a 60-day period to the left metacarpal bones, bored with eight holes of equal diameter and depth, from the middiaphysis toward the distal metaphysis. Eight equal holes bored in the right metacarpal, surrounded by unactivated Helmholtz coils, were taken as controls. The two untreated horses were taken as additional control. The results of computer-assisted histomorphometric analysis indicate that (a) in diaphyseal levels, the amount of bone formed during 60 days is significantly greater (p less than 0.01) in PEMF-treated holes than in contralateral ones and those in control horses; (b) in metaphyseal levels, PEMF-treated holes are sometimes more closed, sometimes less, as compared with contralateral holes and those in control horses; in any case the statistical analysis indicates that the symmetry in the rate of hole repair, found between the two antimeres of control horses, is not appreciable at metaphyseal levels also; (c) there was no statistically significant difference between untreated holes in PEMF-treated horses and holes in control horses, neither at diaphyseal nor at metaphyseal levels. These preliminary findings indicate that PEMFs at low frequency influence the process of bone repair on both diaphysis and metaphysis, and seem to improve the process of bone repair in skeletal regions normally having a lower osteogenetic activity, i.e., in diaphyses as against metaphyses.     

Alcoholism and fracture healing. A radiological study in the rat

Conventional, SPF, and germ-free Wistar rats of both sexes served as laboratory animals. The 2-month-old animals were alcoholized over a period of 1 year using 5%-20% ethanol. The femurs were then osteotomized and treated surgically. Every 2 weeks the course of healing was controlled radiologically. The findings on the process of healing are presented in this work.     

Different healing rates of bone autografts, syngeneic grafts, and allografts in an experimental rat model

Matching of donors and recipients for tissue antigens is vitally important for successful transplantation of essentially all organs and tissues, the major exception being bone. The importance of tissue-typing for the healing of bone allografts remains, however, a controversial issue as development of both humoral and cell-mediated immunity against the grafted bone has been observed in some experimental systems. In the present study, we compared the healing patterns of frozen antigen-mismatched allografts, frozen antigen-matched allografts (syngeneic grafts), and fresh cortical bone autografts in an experimental rat model. Histomorphometry of the graft-host interface revealed that new bone formation started significantly earlier in autografts than in allografts or syngeneic grafts. By 2 weeks, the level of new bone formation in the syngeneic grafts had reached that in autografts. Antigen-mismatched allografts, however, continued to exhibit a retarded formation of new bone throughout the union process. These histomorphometric observations were confirmed by molecular biologic analyses for the mRNA levles of type I collagen, which increased earlier and reached a higher level in autografts than in allografts. Use of syngeneic grafts resulted in a longer persistence of type I collagen mRNA expression in the healing tissue than in antigen-mismatched allografts. No apparent differences were seen between allografts and autografts in the expression of type III collagen. No cartilage-specific type JI collagen mRNA was observed, indicating that antigen-mismatching or preservation by freezing did not alter the basic mechanism of the interface healing process, although it did slow down the beginning of the process. The experiments suggest that a major antigen mismatch between donor and recipient affects the temporal gene expression of extracellular bone matrix and delays new bone formation at the graft-host interface of cortical bone allografts.     

Effects of LLLT in combination with bisphosphonate on bone healing in critical size defects: a histological and histometric study in rat calvaria

The purpose of this study was to analyze histologically the effect of low-level laser therapy (LLLT) in combination with bisphosphonate on bone healing in surgically created critical size defects (CSD) in rat calvaria. One hundred Wistar female rats sham operated (sham) and ovariectomized (Ovx) were maintained untreated for 1 month to allow for the development of osteopenia in the Ovx animals. A CSD was made in the calvarium of each rat, and the animals were divided into five groups according to following treatments: (1) sham rats (control), (2) Ovx rats, (3) Ovx rats treated with LLLT, (4) Ovx rats treated with bisphosphonate, and (5) Ovx rats treated with bisphosphonate and LLLT. Groups 4 and 5 were irrigated with 1 ml of bisphosphonate, and groups 3 and 5 were submitted to LLLT (GaAlAs), 660 nm, 24 J, and 0.4285 W/cm² on the CSD. Ten animals of each treatment were killed at 30 and 60 days. Histomorphometric assessments, using image analysis software, and histological analyses were performed. No defect was completely regenerated with the bone. Histometrically, it can be observed that groups 3 (37.49?±?1.94%, 43.11?±?2.39%) and 5 (35.05?±?1.57%, 41.07?±?1.89%) showed a significant bone neoformation when compared to groups 1 (16.81?±?1.57%, 27.54?±?1.49%), 2 (11.68?±?0.98%, 22.51?±?1.05%), and 4 (14.62?±?1.70%, 25.67?±?1.41%) in all experimental periods (P?<?0.05). It was possible to conclude that the LLLT associated or not with bisphosphonate treatment was effective for stimulating bone formation in CSD in the calvaria of rats submitted to ovariectomy.     

Effects of neonatal estrogenization on rat bone development: A histomorphometric study

Summary The effects of a single dose of 500 μg of estradiol benzoate, administered on the first day of life, on rat bone development have been histomorphometrically studied at 15 days of age. Estrogenized animals presented decreased total tibial length (16.55±0.50 vs. 17.84±0.73 mm,P<0.05) and increased thickness of the cartilage growth plate (528.92±13.30 vs. 382.77±37.85 μm,P<0.01). This increase was mostly due to the presence of a wider (P<0.01) layer of hypertrophic cartilage in the estrogenized rats than in control ones. It might be related to the decreased number of chondroclasts (0.20±0.01 vs. 0.36±0.06 mm⁻¹,P<0.05) found in the resorption zone. Two metaphyseal zones have been considered. In the upper metaphyseal zone there was an increase in the surface density of the cartilaginous trabeculae (49.20±1.80 vs. 40.72±1.95 mm²/mm³,P<0.05), without changes in the volume density. It was related to the presence of thinner and more irregular trabeculae in the estrogenized animals. In the lower metaphyseal zone both the volume (0.19±0.01 vs. 0.14±0.01 mm³/mm³,P<0.01) and surface (34.83±3.01 vs. 26.52±2.46 mm²/mm³,P<0.05) densities of the osseous trabecular tissue were increased in estrogenized rats. No significant differences were found either in the number of osteocytes per area unit of osseous tissue or in the number of osteoclasts per unit length of trabecular osseous tissue.     

Efficacy of contained metaphyseal and periarticular defects treated with two different demineralized bone matrix allografts

The efficacies of two different allografts, Grafton (demineralized bone matrix [DBM] in a glycerol carrier) and Orthoblast (DBM in a reverse thermal poloxamer carrier) were examined from cases involving periarticular fractures. Demographic, perioperative, and outcome data for patients with periarticular fractures who underwent a prospectively designed protocol for bone grafting were compiled, with 15 cases using Orthoblast and 13 using Grafton. A successful graft was defined as healing on the first graft attempt without complications. Healing was determined by radiographic studies and clinical evaluation. The successful graft rates of Orthoblast and Grafton were 15/15 and 9/13, respectively.     

高能量骨折延期手术促进骨愈合的实验研究

目的研究延期手术刺激旺盛的外骨痂生长在高能量骨折中的作用,探索提升骨折愈合能力的新途径。方法取成年狗20只,随机分为ABCD四组,各组均行股骨中段线锯截骨,电凝破坏周围骨膜,制造1 cm缺损,8孔钢板固定。A组截骨14 d后行内固定,B组即时内固定,C组即时内固定,但不用电凝破坏骨膜,D组14 d后行内固定,但固定时切除骨端周围已形成的肉芽。结果狗股骨破坏骨膜制造骨缺损后,早期手术固定组无外骨痂生长,几乎无内骨痂生长,引发了萎缩性骨不连;在同样破坏骨膜制造骨缺损的情况下延期手术固定组产生了旺盛的骨痂生长,产生了稳定固定下的骨痂愈合。结论高能量骨折早期手术固定抑制外骨痂生长,容易造成骨痂生长不良的低质量愈合现象。延期手术固定可以刺激良好的外骨痂生长,改善骨折愈合能力,预防骨不连的发生。     

Peripheral nerve revascularization: histomorphometric study of small- and large-caliber grafts

The revascularization of nerve grafts was investigated using histologic and morphometric techniques. Small-diameter nerve grafts (sciatic in the rat and sural in adult ewes) were studied, as was a large-diameter peroneal nerve graft in the ewe. Ninety-six hours after sciatic nerve engraftment, rats were injected with an intravascular fluorescent tracer. Evans blue albumin (EBA). Specimens were observed for the number of vessels perfused. Analysis showed no difference in vascular pattern between the grafted nerves and their control nerves, suggesting that spontaneous revascularization had occurred to establish a vascular tree essentially identical to the native nerve. Sural and peroneal nerve grafts were evaluated in adult ewes at 7 or 40 days post-nerve grafting. Similar to the rat sciatic nerve, the small-diameter sural nerve grafts were completely revascularized, with an equal number of perfused vessels at both time periods, with respect to control specimens. In contrast, the larger-caliber peroneal nerve grafts were not perfused at 7 days, and very poorly perfused at 40 days. This correlated with scant neural regeneration at 40 days. The finding suggests that small-diameter nerve grafts spontaneously revascularize, and revascularization using microvascular techniques is not necessary. In contrast, the larger-diameter nerve graft did not revascularize well. Such a large-diameter nerve graft would provide a suitable model to investigate the potential merits of a vascularized nerve graft.     

The effect of ethanol intake on tendon healing: a histological and biomechanical study in a rat model

Introduction  

Ethanol has a suppressive effect on inflammation and the immune system, but the effect of ethanol on tendon healing in vivo has not been studied. The purpose of this study was to investigate the histological and biomechanical effects of ethanol intake on tendon healing in a rat tendon injury model.