Effect of diffusion chamber pore size on differentiation and proliferation of periosteal cells. An experimental study

The influence of the pore size of Nucleopore diffusion-chamber filters on the rate of proliferation and differentiation of periosteal cells in muscle was studied in 44 growing rabbits. Periosteal grafts were placed in chambers (16-19 in each experimental group) sealed with filters with a pore size of 0.4, 0.6, 0.8, 1.0, or 2.0 micron. Each chamber was implanted into the paraspinal muscle of the rabbit, where it remained for 16 weeks. The osteochondrogenic activity of the graft grew linearly when the pore size increased from 0.4 to 1.0 micron. In the chambers with a pore size of 2.0 micron, both bone and cartilage were found in only one chamber. Bone and cartilage were not found outside any of the chambers. The present results showed that the pore size of the filters significantly affected the ability of the periosteal graft to form bone and cartilage.     

Histology and histomorphometry of bone regeneration after experimental injuries.

Reparative callus formation upon tubular bone was studied after surgical injuries of different degrees. Thirty-seven young rats were divided into three groups. In the periosteum group the bone was scraped, in the fissure group we made a slit reaching the medulla, and in the defect group a standard defect was sawn. Rats were killed at 6, 12 and 18 days. The formed callus was studied histologically and histomorphometrically. The results suggest than even the primary osteochondrogenic callus formation is dependent on the mode of injury. At six days the three groups differed both qualitatively and quantitatively in regard to callus formation. At 12 and 18 days the total area of woven bone was proportional to the degree of trauma according to the linear regression line Y = 0.883 x X + 0.226 (r = 0.876, P less than 0.001). In the fissure and defect groups reparative bone filled the hole proportionally to the same extent. Periosteal and endosteal woven bone formed round the bone. Medullary bone formation was limited mainly to the area immediately adjacent to the trauma, the result being a cylinder-shaped callus. Cartilage formation was most abundant at six days and was related to the amount of woven bone in the external callus (r = 0.854, P less than 0.001) and to the trauma area (r = 0.707, P less than 0.05).     

Qualitative and quantitative analysis of orthotopic bone regeneration by marrow

A rat model of a femoral segmental defect was used to specifically test the hypothesis that autogenous marrow has the osteogenic capability to heal a bone defect. The variables analyzed included the ratio of the marrow volume to the defect, implantation of live or dead marrow, and remodeling of established nonunions by implantation of live marrow. The uniqueness of this model allows biomechanical evaluation of the new bone formed by the implant. When live marrow was implanted, woven bone formed at 3 weeks, progressing to early lamellar bone at 6 weeks, with subsequent remodeling for as long as 12 weeks in a volumetric fashion (p < 0.05). Bone marrow, when placed in a fresh femoral defect and given in sufficient amounts, produced a rate of union comparable with that of autologous bone grafts. Mature lamellar bone formed by marrow was evaluated biomechanically: the results were statistically comparable with those of cancellous bone grafts at 12 weeks. Significant bone formation occurred when marrow was percutaneously injected in femoral nonunions, although union and remodeling did not take place in this rat model. Implantation of dead marrow resulted in rare cellular infiltration and minimal bone formation in a manner comparable with that of autogenous cancellous bone grafts. These results indicate that bone marrow can lead to structurally functional bone regeneration in an orthotopic location.     

Bone graft incorporation. Effects of osteogenic protein-1 and impaction

Impaction of cancellous bone grafts in a bone chamber in rats in a previous study led to decreased ingrowth of new bone after 6 weeks compared with unimpacted grafts. The current study analyzes whether this decrease represented a final loss of ingrowth or just a delay, if the decrease was influenced by immunologic factors, and if it was possible to influence the inhibitory effect by adding a bone morphogenetic protein. Bone chambers with impacted or unimpacted bone grafts were implanted bilaterally in rat tibias. The mean bone ingrowth distance into the graft was measured on histologic sections. Three experiments were done: (1) the bone ingrowth into impacted and unimpacted grafts was studied at 6 and 12 weeks; (2) the immunologic influence was studied by comparing isogeneic grafts with allogeneic grafts; and (3) the authors tried to influence the decrease in bone ingrowth in impacted grafts by adding osteogenic protein-1. Bone ingrowth into the impacted graft was decreased at 6 weeks but not at 12 weeks. No difference was found between isografts and allografts at 6 weeks. With the addition of osteogenic protein-1, the impacted grafts showed dramatically increased bone ingrowth. Impacted bone grafts are incorporated at a slower rate than were structural grafts. The delay can be reversed by adding osteogenic protein-1, making ingrowth faster than in structural bone.     

Healing course of primate ulna segmental defects treated with osteogenic protein-1.

Twelve African green monkeys were implanted with recombinant human osteogenic protein-1 (rhOP-1) placed on a bovine bone-derived Type I collagen carrier to characterize healing in an ulna segmental bone defect model at 1, 3, 12, and 20 weeks postoperative. Defect healing was evaluated by plain film radiography, computed tomography (CT), magnetic resonance imaging (MRI), bone mineral density (BMD), and histologic analysis. Radiographically, new bone formation was observed as early as 3 weeks postoperative. By 6 weeks, new bone was visible in five of six defects. Increased quantity and mineralization of the new bone were apparent by 12 weeks. Reformation of the medullary cavity with appearance of marrow elements was demonstrated by CT and MRI at 20 weeks. BMD studies revealed a significant increase in the presence of bone with time. Histology at 1 week demonstrated that the implant material was well contained in the defect, and a proliferation of cells occurred at the defect borders. At 3 weeks cell proliferation continued and cell phenotype differentiation was recognized. By 12 weeks substantially less residual carrier was found in the defects, and calcifying tissues with plump chondrocytes, osteoblasts, and immature woven bone were observed. Areas of lamellar and woven bone were identified at 12 weeks, with advanced remodeling and revascularization observed at 20 weeks. The use of osteoinductive implants may provide an alternative to autologous and allogeneic bone tissue in the therapeutic approach to bone defects and promotion of fusion by eliminating the donor site morbidity associated with autogenous bone and the decreased efficacy and potential for disease transmission associated with allogeneic bone.     

Ivan Petrovich Pavlov

Twelve African green monkeys were implanted with recombinant human osteogenic protein-1 (rhOP-1) placed on a bovine bone-derived Type I collagen carrier to characterize healing in an ulna segmental bone defect model at 1, 3, 12, and 20 weeks postoperative. Defect healing was evaluated by plain film radiography, computed tomography (CT), magnetic resonance imaging (MRI), bone mineral density (BMD), and histologic analysis. Radiographically, new bone formation was observed as early as 3 weeks postoperative. By 6 weeks, new bone was visible in five of six defects. Increased quantity and mineralization of the new bone were apparent by 12 weeks. Reformation of the medullary cavity with appearance of marrow elements was demonstrated by CT and MRI at 20 weeks. BMD studies revealed a significant increase in the presence of bone with time. Histology at 1 week demonstrated that the implant material was well contained in the defect, and a proliferation of cells occurred at the defect borders. At 3 weeks cell proliferation continued and cell phenotype differentiation was recognized. By 12 weeks substantially less residual carrier was found in the defects, and calcifying tissues with plump chondrocytes, osteoblasts, and immature woven bone were observed. Areas of lamellar and woven bone were identified at 12 weeks, with advanced remodeling and revascularization observed at 20 weeks. The use of osteoinductive implants may provide an alternative to autologous and allogeneic bone tissue in the therapeutic approach to bone defects and promotion of fusion by eliminating the donor site morbidity associated with autogenous bone and the decreased efficacy and potential for disease transmission associated with allogeneic bone.     

Regeneration of Defects in Articular Cartilage with Callus and Cortical Bone Grafts an experimental study

Cartilage regeneration was studied in an experiment in rats. A standardised full-thickness articular cartilage defect was created and autogenous 12-day-old callus or cortical bone graft was transplanted into it, or the defect was left empty. The follow up periods were three, six, 12, and 24 weeks, and each subgroup consisted of five animals. A total of 60 animals were operated on. From six weeks onwards hyaline-like cartilaginous tissue had started to develop at the edges of the defect in all three groups. In the middle section of the hole, however, the picture was different; at 24 weeks none of the specimens in the defect group, two of the five in the callus graft group, and all five in the bone graft group had developed full-thickness, hyaline-like cartilaginous regeneration. The hyaline-like cartilaginous tissue in the medial segment was hypocellular when analysed by histomorphometry. On scanning electron microscopy the surface of the reparative tissue looked fibrillated in all specimens from the three groups.     

Bone repair induced by bone morphogenetic protein in ulnar defects in dogs

In dogs, resection of a length of the ulna equal to twice the diameter of the mid-shaft leaves a defect which consistently fails to unite. In response to an implant of 100 mg of bovine bone morphogenetic protein (BMP), the defect becomes filled by callus consisting of fibrocartilage, cartilage and woven bone within four weeks. The cartilage is resorbed and replaced by new bone in four to eight weeks. Woven bone is then resorbed, colonised by bone marrow cells and remodelled into lamellar bone. Union of the defect is produced by 12 weeks. Control defects filled with autogeneic cortical bone chips unite after the same period. In regeneration induced by bone morphogenetic protein (BMP) and in repair enhanced by bone graft, union depends upon the proliferation of cells within and around the bone ends. Our working hypothesis is that BMP induces the differentiation of perivascular connective tissue cells into chondroblasts and osteoprogenitor cells and thereby augments the process of bone regeneration from the cells already present in the endosteum and periosteum.     

Repair of segmental bone defects in the rat: an experimental model of human fracture healing.

Bone repair models in animals may be considered relevant to human fracture healing to the extent that the sequence of events in the repair process in the model reflect the human fracture healing sequence. In the present study, the relevance of a recently developed segmental defect model in rat fibula to human fracture healing was investigated by evaluating temporal progression of rigidity of the fibula, mineral content of the repair site, and histological changes. In this model, a surgically created 2-mm-long defect was grafted with a 5-mm-long tubular specimen of demineralized bone matrix (DBM) by inserting it over the cut ends of the fibula. The temporal increase in rigidity of the healing fibula demonstrated a pattern similar to biomechanical healing curves measured in human fracture healing. This pattern was characterized by a short phase of rapidly rising rigidity during weeks 4-7 after surgery, associated with a sharp increase in the mineral content of the repair tissue. This was preceded by a phase of nearly zero rigidity and followed by a phase of slow rate of increase approaching a plateau. Histologically, chondroblastic and osteoblastic blastema originating from extraskeletal and subperiosteal (near fibula-graft junction) regions, infiltrated the DBM graft during the first 2 weeks. The DBM graft assumed the role of a "bridging callus." By weeks 6-8, most of the DBM was converted to new woven and trabecular bone with maximal osteoblastic activity and minimal endochondral ossification. Medullary callus formation started with direct new bone formation adjacent to the cortical and endosteal surfaces in the defect and undifferentiated cells in the center of the defect at 3 weeks. The usual bone repair process in rodents was altered by the presence of the DBM graft to recapitulate the sequential stages of human fracture healing, including the formation of a medullary callus, union with woven and lamellar bone, and recreation of the medullary canal.     

Callus formation after re-injury to experimental bone defect

Summary Osteochondrogenic callus formation after delayed re-injury at different time intervals was evaluated. Twenty young rats were operated, and a standard bone defect reaching the medulla was sawn into the left hind leg. The rats were divided into two groups and the defect was re-created after 4 or 12 days. The rats were killed 7 or 14 days after the second operation. The developing new bone was revealed with a triple fluorochrome labelling system. The areas of bone and cartilage were measured histomorphometrically. The results suggested that an external factor, re-trauma, could increase both external and internal primary osteogenic callus formation. The optimum time to potentiate bone formation in the defects was found to be earlier than identifed in previous fracture studies. After a 4-day delay the external callus was significantly larger than after the 12-day delay. Cartilage formation was not enhanced despite the sudden increase in osteogenic callus formation.     

Time-dependent sensory nerve ingrowth into a bone conduction chamber

We studied time-dependent ingrowth of sensory nerve fibers into a bone defect in a rat bone conduction chamber model. In 10 male Sprague Dawley rats, a titanium chamber was implanted bilaterally in the proximal tibiae, representing an experimental bone defect. To mimic a clinical situation, the chambers were filled with a fresh blood clot. After 1, 2, 4, 6 and 8 weeks, 2 rats were fixed in vivo at each time before removal of specimens, and histological and immunohistochemical analyses. We used antisera against protein gene product 9.5, neural growth-associated protein 43/B-50, calcitonin gene-related peptide, and substance P, to locate regenerating sensory nerve fibers in the chamber. During bone defect healing, hematoxylin/eosin sections showed that new bone grew in through the ingrowth openings in the chamber, gradually filling it and replacing the blood clot. At 1 and 2 weeks after implantation, no nerve fibers could be detected. At 4, 6 and 8 weeks, however, small numbers of nerve fibers were seen in 8 of 11 specimens. The nerve fibers were located mainly in the dense fibrous tissue in close proximity to the new bone, and in some cases within the new forming bone. In this chamber model, the periosteum is not in contact with the bone ingrowth openings, and all ingrowing nerve fibers thus originated from the cortical bone, endosteum or bone marrow. We speculated that these late ingrowing sensory nerve fibers may actively participate in bone repair.     

Time-dependent sensory nerve ingrowth into a bone conduction chamber

We studied time-dependent ingrowth of sensory nerve fibers into a bone defect in a rat bone conduction chamber model. In 10 male Sprague Dawley rats, a titanium chamber was implanted bilaterally in the proximal tibiae, representing an experimental bone defect. To mimic a clinical situation, the chambers were filled with a fresh blood clot. After 1, 2, 4, 6 and 8 weeks, 2 rats were fixed in vivo at each time before removal of specimens, and histological and immunohistochemical analyses. We used antisera against protein gene product 9.5, neural growth-associated protein 43/B-50, calcitonin gene-related peptide, and substance P, to locate regenerating sensory nerve fibers in the chamber. During bone defect healing, hematoxylin/eosin sections showed that new bone grew in through the ingrowth openings in the chamber, gradually filling it and replacing the blood clot. At 1 and 2 weeks after implantation, no nerve fibers could be detected. At 4, 6 and 8 weeks, however, small numbers of nerve fibers were seen in 8 of 11 specimens. The nerve fibers were located mainly in the dense fibrous tissue in close proximity to the new bone, and in some cases within the new forming bone. In this chamber model, the periosteum is not in contact with the bone ingrowth openings, and all ingrowing nerve fibers thus originated from the cortical bone, endosteum or bone marrow. We speculated that these late ingrowing sensory nerve fibers may actively participate in bone repair.     

Time-dependent sensory nerve ingrowth into a bone conduction chamber

We studied time-dependent ingrowth of sensory nerve fibers into a bone defect in a rat bone conduction chamber model. In 10 male Sprague Dawley rats, a titanium chamber was implanted bilaterally in the proximal tibiae, representing an experimental bone defect. To mimic a clinical situation, the chambers were filled with a fresh blood clot After 1, 2, 4, 6 and 8 weeks, 2 rats were fixed in vivo at each time before removal of specimens, and histological and immunohistochemical analyses. We used antisera against protein gene product 9.5, neural growth-associated protein 43/B-50, calcitonin gene-related peptide, and substance P, to locate regenerating sensory nerve fibers in the chamber. During bone defect healing, hematoxylin/eosin sections showed that new bone grew in through the ingrowth openings in the chamber, gradually filling it and replacing the blood clot. At 1 and 2 weeks after implantation, no nerve fibers could be detected. At 4, 6 and 8 weeks, however, small numbers of nerve fibers were seen in 8 of 11 specimens. The nerve fibers were located mainly in the dense fibrous tissue in close proximity to the new bone, and in some cases within the new forming bone. In this chamber model, the periosteum is not in contact with the bone ingrowth openings, and all ingrowing nerve fibers thus originated from the cortical bone, endosteum or bone marrow. We speculated that these late ingrowing sensory nerve fibers may actively participate in bone repair.     

Differentiation of periosteal cells in muscle. An experimental study using the diffusion chamber method

The osteo-chondrogenic capacity of the undifferentiated mesenchymal cells of the periosteum has been made use of in clinical reconstructive surgery. In the present investigation we studied the osteo-chondrogenic potency of free periosteal transplants in muscle using the diffusion chamber method. A total of 42 experimental and seven control rabbits aged four to six weeks were operated on. Periosteum was obtained from the anterior aspect of the left tibial bone by stripping. The grafts were placed in Nucleopore diffusion chambers with a pore size of 0.4 micron. The chambers were implanted in the anterior tibial and paraspinal muscles of the rabbit. Osteogenesis began after the second postoperative week and increased up to the 5-6 week interval when a plateau phase was reached. Chondrogenesis, which also began after the second postoperative week, reached two plateau phases; the first observed at 4-8 weeks and the second at 12-16 weeks. Neither bone nor cartilage formation could be observed outside the chambers. In the semi-open control chambers with only one end sealed, bone formed within the chamber as early as two weeks after transplantation and grew out into the adjacent connective tissue of muscle. It is noteworthy that the periosteal transplant retained its osteochondrogenic properties even when isolated in the diffusion chamber. The young age of the donor animals might have contributed to our findings.     

不同长度骨缺损模型中皮质骨内变化及作用

目的：本文应用非脱钙骨切片技术研究皮质骨在1mm和10mm骨缺损修复过程中的变化及其在骨修复中的作用。方法：新西兰大白兔30只，分成1、2、3、4、6周组，每组再分成1mm及10mm2组，每组3只，右侧分别为1、10mm骨缺损侧，左侧为对照。制作非脱钙骨切片，组织学观察骨缺损愈合过程中皮质骨结构变化。结果：组织学观察显示，两骨缺损模型修复过程的早期，皮质骨断端各表面均出现了骨融解现象，皮质骨陷窝内的骨细胞被溶解并释放出，与周围骨缺损修复组织联系紧密。4周后，在1mm骨缺损模型中，在外骨痂附近的皮质骨部位有许多的改建与再建的哈佛氏系统单位，皮质骨内的再建结构与外骨痂及连接骨痂内的再建结构紧密相连；而10mm模型中，皮质骨无明显变化。结论：伴随骨修复过程，骨缺损两端皮质骨发生特征性改变，在骨愈合再生期，可为骨修复过程提供促骨修复细胞及物质；而在骨愈合的改建期，也参与各种骨痂的改建过程。因此，皮质骨是骨修复过程的再生及重建过程中重要影响因素。     

Osteochondrogenesis of Free Periosteal Grafts in the Rabbit Iliac Crest

The influence of the bone marrow, cortical bone and apophyseal cartilage of the iliac crest on osteochondrogenesis from free autogenous periosteal grafts was studied histologically in 8-week-old rabbits. Tibial periosteum was transplanted around the iliac crest, from which the periosteum had been removed from the inner side, periosteum and cortical bone in an area on the outer side and perichondrium from the apophyseal cartilage. Most bone formation occurred in the area with periosteum in contact with the bone marrow of the cancellous bone. By means of an isolating Nucleopore filter^®, it was revealed that the most vigorous of this bone formation originated from the periosteal graft. Further, it was noted that in the series with the Nucleopore filter, bone formation was slower than in the series without the filter, suggesting some inductive factors. No bone formation occurred in the apophyseal area.     

Human parathyroid hormone (1-34) accelerates the fracture healing process of woven to lamellar bone replacement and new cortical shell formation in rat femora

This study aimed to test whether intermittent treatment of human parathyroid hormone [hPTH(1-34)] disturbs or accelerates the fracture healing process using rat surgical osteotomy model. One hundred five, 5-week-old SD rats were allocated to vehicle control (CNT) and four PTH groups; 10 and 30 microg/kg of hPTH(1-34) treatment before surgery (P10, P30), and treatment before and after surgery (C10, C30). All animals were given subcutaneous injections three times a week for 3 weeks. Then, fractures were produced by transversely cutting the midshaft of bilateral femora and fixing with intramedullary wire. Human PTH(1-34) treatment was continued in C10 and C30 groups until sacrifice at 3, 6, and 12 weeks after surgery. The femora were assessed by peripheral quantitative computed tomography, three-point bending mechanical test, and histomorphometry. Total cross-sectional area was not significantly different among all groups at any time point. At 3 weeks after surgery, the lamellar bone/callus area was significantly increased in C10 and C30 groups compared to the other groups. At 6 weeks, remodeling of woven bone to lamellar bone in the callus was almost complete in all groups. At 12 weeks, percent new cortical shell area was significantly higher in C10 and C30 groups compared to the other groups, and the ultimate load in mechanical testing was significantly higher in C30 group than in CNT, P10, and P30 groups. Intermittent PTH treatment at 30 microg/kg before and after osteotomy accelerated the healing process as evidenced by earlier replacement of woven bone to lamellar bone, increased new cortical shell formation, and increased the ultimate load up to 12 weeks after osteotomy.     

Mechanical loading enhances the anabolic effects of intermittent parathyroid hormone (1-34) on trabecular and cortical bone in mice

The separate and combined effects of intermittent parathyroid hormone (iPTH) (1-34) and mechanical loading were assessed at trabecular and cortical sites of mouse long bones. Female C57BL/6 mice from 13 to 19 weeks of age were given daily injections of vehicle or PTH (1-34) at low (20 microg/kg/day), medium (40 microg/kg/day) or high (80 microg/kg/day) dose. For three alternate days per week during the last two weeks of this treatment, the tibiae and ulnae on one side were subjected to a single period of non-invasive, dynamic axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle). Two levels of peak load were used; one sufficient to engender an osteogenic response, and the other insufficient to do so. The whole tibiae and ulnae were analyzed post-mortem by micro-computed tomography with a resolution of 5 microm. Treatment with iPTH (1-34) modified bone structure in a dose- and time-dependent manner, which was particularly evident in the trabecular region of the proximal tibia. In the tibia, loading at a level sufficient by itself to stimulate osteogenesis produced an osteogenic response in the low-dose iPTH (1-34)-treated trabecular bone and in the proximal and middle cortical bone treated with all doses of iPTH (1-34). In the ulna, loading at a level that did not by itself stimulate osteogenesis was osteogenic at the distal site when combined with high-dose iPTH (1-34). At both levels of loading, there were synergistic effects in cortical bone volume of the proximal tibia and distal ulna between loading and high-dose iPTH (1-34). Images of fluorescently labelled bones confirmed that such synergism resulted from increases in both endosteal and periosteal bone formation. No woven bone was induced by iPTH (1-34) or either level of loading alone, whereas the combination of iPTH (1-34) and the "sufficient" level of loading stimulated woven bone formation on endosteal and periosteal surfaces of the proximal cortex in the tibiae. Together, these data suggest that in female C57BL/6 mice, under some but not all circumstances, mechanical loading exerts an osteogenic response with iPTH (1-34) in trabecular and cortical bone.     

The Otto Aufranc Award. Strut allograft healing to the femur with recombinant human osteogenic protein-1

Allograft struts are used to reinforce the deficient proximal femur in hip arthroplasty or for fixation of a periprosthetic fracture. Although the use of strut grafts wired or cabled to the proximal femur generally has been successful, the time for healing is slow. The purpose of the current study was to determine whether cortical strut graft healing to the femur could be enhanced by the addition of recombinant human osteogenic protein-1. Fourteen adult dogs underwent bilateral onlay allograft strut procedures to the midfemur using stainless steel cables. In each animal one femur received 500 mg of osteogenic protein-1 device (2.5 mg recombinant human osteogenic protein-1/g Type I collagen) interposed between the graft and host bone. The results showed that the healing of cortical strut grafts to the femur was enhanced dramatically by the addition of the osteogenic protein-1 device. The sites treated with osteogenic protein-1 had significantly greater radiographic, histologic, and microradiographic scores at all times. Rapid formation of new bone and graft incorporation was observed in sites treated with the osteogenic protein-1 device. Strut healing with the osteogenic protein-1 device at 4 weeks postoperative was superior to the healing in control sites at 8 weeks. Improving and accelerating the course of cortical strut graft healing should provide a substantial clinical benefit in lowering the risk of graft nonunion and fracture and shorten the time of protected weightbearing and functional disability.     

Study of the biomechanical characteristics of fracture callus

For the purpose of clarifying the fracture healing process from a rheological standpoint, the author attempted to study the change in physical characteristics of the callus. The study was conducted as follows: a cortical defect without fracture was made in the tibia of a mature rabbit, and a dynamic viscoelasticity test and static tensile test were made. A two-dimensional finite element model of the fracture area of the long bone was developed to simulate changes in the strength. Thus, the difference in the strength of the fracture area with and without internal fixation was compared and studied. As results, 80% of the strength was gained within 12 weeks in both the dynamic viscoelasticity test and the static tensile test. It also became clear that the bending strength of non-fixation group with an abundant external callus exceeded that of the internal fixation extraction group in the simulation test.