Cellular origin of endochondral ossification from grafted periosteum

Grafted periosteum is known to have potential for heterotopic bone formation by endochondral ossification. Although osteochondrogenic cells have been thought to originate from the osteogenic layer in grafted periosteum, no histological report has yet demonstrated this. The present study was designed to elucidate the origin of chondrogenesis preceding bone formation in grafted periosteum. Periostea harvested from young Japanese white rabbits' tibiae were grafted into suprahyoid muscles and examined radiographically and histologically at postoperative days 1, 7, 9, 14, 21, and 35. Normal periostea and tibial graft site were also examined. Surgical harvesting of the periosteum split and damaged its osteogenic layer but retained the fibrous layer intact. Most of the osteoblasts remained on the tibial bone surface, and only few cells of the osteogenic layer were present in grafted tissue. By the seventh day after grafting, the fibrous layer had thickened. The fibroblastic cells in the fibrous layer had significantly increased in number (P < 0.01) and were positively stained for proliferating cell nuclear antigen. These cells exhibited alkaline phosphatase activity at day 9. The differentiated chondrocytes had formed cartilage at postoperative day 14. Cells in the osteogenic layer appeared necrotic and subsequently disappeared. Following postoperative day 21, cartilage was replaced by trabecular bone. Bone formation was completed by 35 days. An X‐ray analysis at this time also revealed new bone formation. These findings indicate that grafted periosteum forms bone by endochondral ossification and that the cells of the fibrous layer play essential roles in chondrogenesis that precedes such bone formation. Anat Rec 264:348–357, 2001. © 2001 Wiley‐Liss, Inc.     

Calcium sulfate-carboxymethylcellulose bone graft binder: Histologic and morphometric evaluation in a critical size defect

Calcium sulfate (CS) is widely used as a bone graft binder and expander. Recent reports indicate that carboxymethylcellulose (CMC) can improve the clinical properties of CS when used as binder for particulate bone grafts; however, limited information is available on the effects of CMC on bone regeneration. The purpose of this study was to evaluate the histologic and morphometric characteristics of bone formation in calvarial defects grafted with a CS-based putty containing 10% CMC in combination with allogeneic demineralized bone matrix (DBM). Bone formation and graft/binder resorption were compared with a surgical grade CS and DBM in paired critical-sized calvarial defects in 25 Wistar rats (350-450 g). Six animals each provided paired defects at 7, 14, 21, and 28 days postsurgery for nondecalcified processing and microscopic analysis. Defects grafted with CS or CS-CMC putty as the DBM binder exhibited similar patterns and proportions of bone formation, fibrous tissue/marrow, and residual DBM particles. Comparable mean +/- SD proportions of new bone formation (31.7 +/- 9.5 and 33.7 +/- 12.9), fibrous tissue/marrow (54.2 +/- 8.3 and 53.0 +/- 10.8), residual DBM particles (8.3 +/- 6.8 and 10.1 +/- 6.3), and residual binder material (5.5 +/- 4.6 and 3.7 +/- 3.5) were found at 28 days for defects grafted with CS and CS-CMC putty, respectively. Thus, CMC was found to improve the handling characteristics of CS and, when used in conjunction with DBM, supported comparable levels bone formation and patterns of binder/scaffold resorption as CS and DBM in a calvarial defect model.     

The influence of osteoblast differentiation stage on bone formation in autogenously implanted cell-based poly(lactide-co-glycolide) and calcium phosphate constructs

We tested the hypothesis that the osteoblast differentiation status of bone marrow stem cells (BMSCs) combined with a three-dimensional (3D) structure modulates bone formation when autogenously implanted. Rat BMSCs were aspirated, expanded, and seeded into a 3D composite of poly(lactide-co-glycolide) and calcium phosphate (PLGA/CaP) to produce a hybrid biomaterial. Calvarial defects were implanted with (1) scaffold without cells (SC/NC), (2) scaffold and BMSCs (SC+BMSC), (3) scaffold and osteoblasts differentiated for 7 days (SC+OB7), and (4) for 14 days (SC+OB14). After 4 weeks, there was more bone formation in groups combining scaffold and cells, SC+BMSC and SC+OB7. A nonsignificant higher amount of bone formation was observed on SC+OB14 compared with SC/NC. Additionally, more blood vessels were counted within all hybrid biomaterials, without differences among them, than into SC/NC. These findings provide evidences that the cell differentiation status affects in vivo bone formation in autogenously implanted cell-based constructs. Undifferentiated BMSCs or osteoblasts in early stage of differentiation combined with PLGA/CaP scaffold favored bone formation compared with plain scaffold and that one associated with more mature osteoblasts.     

Bone grafts cultured with bone marrow stromal cells for the repair of critical bone defects: an experimental study in mice

Tissue engineering of autologous bone combined with osteoprogenitor cells is a suitable strategy for filling large bone defects. The aim of this study was to evaluate the osteogenicity of a xenogenic bone graft cultured with allogenic bone marrow stromal cells (BMSC) in a mouse critical size craniotomy. Bovine trabecular bone grafts were made free of bone marrow cells or debris and were delipidated. BMSC were harvested from C57BL/6-Tg(ACTbEGFP)1Osb/J mice (GFP+ cells) and were cultured 14 days on bone grafts in control or osteogenic medium. Engineered grafts were implanted in calvarial defect in C57BL/6 mice. Four groups were studied: graft with BMSC differentiated in osteoblasts (G-Ob), graft with BMSC (G-BMSC), graft without cells (G) and no graft. Calvariae were studied 2 and 8 weeks after implantation by radiographic and histomorphometric analyses. G group: the bone ingrowth was limited to the edges of the defect. The center of the graft was filled by a fibrovascular connective tissue. G-BMSC or G-Ob groups: bone formation occurred early in the center of the defect and did not increase between 2 and 8 weeks; the newly formed woven bone was partially replaced by lamellar bone. The preoperative osteoblastic differentiation of BMSC did not allow faster and better bone regeneration. After 2 weeks, GFP+ cells were observed around the grafted bone but no GFP+ osteocyte was present in the newly formed bone. No GFP+ cell was noted after 8 weeks. However, pre-implantation culture of the biomaterial with allogenic BMSC greatly enhanced the bone regeneration.     

Cellular origin of endochondral ossification from grafted periosteum.

Grafted periosteum is known to have potential for heterotopic bone formation by endochondral ossification. Although osteochondrogenic cells have been thought to originate from the osteogenic layer in grafted periosteum, no histological report has yet demonstrated this. The present study was designed to elucidate the origin of chondrogenesis preceding bone formation in grafted periosteum. Periostea harvested from young Japanese white rabbits' tibiae were grafted into suprahyoid muscles and examined radiographically and histologically at postoperative days 1, 7, 9, 14, 21, and 35. Normal periostea and tibial graft site were also examined. Surgical harvesting of the periosteum split and damaged its osteogenic layer but retained the fibrous layer intact. Most of the osteoblasts remained on the tibial bone surface, and only few cells of the osteogenic layer were present in grafted tissue. By the seventh day after grafting, the fibrous layer had thickened. The fibroblastic cells in the fibrous layer had significantly increased in number (P < 0.01) and were positively stained for proliferating cell nuclear antigen. These cells exhibited alkaline phosphatase activity at day 9. The differentiated chondrocytes had formed cartilage at postoperative day 14. Cells in the osteogenic layer appeared necrotic and subsequently disappeared. Following postoperative day 21, cartilage was replaced by trabecular bone. Bone formation was completed by 35 days. An X-ray analysis at this time also revealed new bone formation. These findings indicate that grafted periosteum forms bone by endochondral ossification and that the cells of the fibrous layer play essential roles in chondrogenesis that precedes such bone formation.     

Effects of periosteal activation agent on bone repair and ingrowth

A substance that activates the resting periosteum (PAA) was applied to the periosteal surface in two different healing models using the femurs of 2-kg male rabbits. The activation agent was applied to the periosteal surface over the sites of circular defects drilled through the lateral cortex in one model and over the sites of porous polyethylene implants placed in the lateral cortex and the medullary canal in the other model. Results failed to show that the agent either enhanced bone ingrowth into the porous implants or accelerated bony filling of the circular defects. However, there was indication of enhanced mineralization and periosteal callus formation as early as 24 h after application.     

Osteoblast and osteoclast precursors in primary cultures of calvarial bone cells

Bone cells obtained by digestion of fetal mouse or chicken calvaria were tested for their ability to form or resorb bone in vitro. The isolated cells were precultured for 6 days and subsequently cocultured for 11 days with periosteum-free noninvaded fetal mouse long bone rudiments. Bone formation and resorption during coculture were evaluated by histology and 45Ca release from prelabeled bones. The calvarial origin of cells in cocultures was traced by labeling the cells with 3H-thymidine before coculture, followed by autoradiography. Many osteoblasts and osteoclasts as well as fibroblasts developed from mouse periosteal cells released late in the sequential digestion procedure and previously denoted as "osteoblastlike" (BL). No or few osteoblasts and osteoclasts but many fibroblasts developed from early released cell fractions that have previously been denoted as "osteoclastlike" (CL). Only osteoblasts and fibroblasts but not osteoclasts developed from chicken calvarial cell fractions. The osteoblasts developed primarily from cell fractions from the inner layer of the periosteum, previously denoted as "osteoblastlike" (OB). Cells obtained from the outer layer of the periosteum (PF) gave rise mainly to fibroblasts. These studies show that osteoblast and osteoclast precursor cells are maintained in monolayer cultures of periosteal cell fractions. However, sequential digestion of mouse calvaria does not lead to separation of the two types of bone cells. Rather, osteoclast and osteoblast precursors are released jointly, from the periosteal cell layers closest to the bone surface. In the chicken cell fractions osteoclast precursors are absent after preculture, resulting in a more homogeneous population of osteoblast and fibroblast but not osteoclast precursors.     

In vivo model for frontal sinus and calvarial bone defect obliteration with bioactive glass S53P4 and hydroxyapatite

An in vivo model was developed to investigate the usability of a frontal sinus and a calvarial bone defect obliteration with bioactive glass S53P4 (BG) and hydroxyapatite (HA) granules. Roofs of 21 Elco rabbit frontal sinuses were drilled open from 4 separate holes using a standard method, and the sinuses, located in pairs, in frontal bone were filled with BG on one side and with HA on the other side. Two parallel posterior defects were covered with a pedicled periosteum flap, and 2 anterior defects with a free flap. The stability of materials, new bone, and connective tissue formation were observed with histomorphometry, scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDXA), and X-ray pictures at 1, 3, 6, and 12 months postoperatively. The results showed more rapid resorption of filling material (p = 0.019) and new bone formation (p = 0.0001) in the defects filled with BG than in the corresponding HA-filled defects studied by histomorphometry throughout the study. New bone formation and resorption of materials were faster in defects covered by a pedicled flap than by a free periosteum flap. The results were supported by SEM histomorphometric and radiologic analysis. Both bioactive materials studied were well tolerated in frontal sinuses and in calvarial bone defects. The experimental model showed the influence of early periosteum vascularization on accurate frontal sinus filling and the healing process in rabbit frontal sinuses.     

Evaluation of bone regeneration at critical-sized calvarial defect by DBM/AM composite

This study investigated the bone-regenerative potential of a demineralized bone and acellular matrix (DBM/AM) composite (AlloCraft DBM) in comparison with autologous bone using an in vivo model. Critical-sized calvarial defects (5 mm) were created in athymic rats. The defects were grafted with either the DBM/AM composite or the acellular human dermal matrix (AM), and compared with the defects filled with autologous bone (positive control) and the empty defect (negative control). Histological and radiographic assessments were carried out at 4 and 8 weeks after surgery to determine the biological healing, the amount and type of new bone formation and the percentage of new bone filled in the critical defects. At 4 weeks, DBM/AM composite group had the highest percentage of the defect filled with new bone (84%), which was significantly greater than autologous bone (62%), AM (41%), and untreated control (32%) groups. At 8 weeks, the DBM/AM continued to have the highest percentage of the defect filled with new bone (91%). The autologous bone group increased the percentage of bone fill to 83%. The defects either filled with AM or left untreated still had less of the defect filled with new bone, 57% and 33%, respectively. The total healing of defects grafted with DBM/AM was comparable with autologous bone group at 8 weeks. The results demonstrated that the DBM/AM composite promoted new bone formation more rapidly than autologous bone at calvarial defect in athymic rats. The study supports that DBM/AM is a potential substitute of autologous bone for bone repair.     

The fate of allogeneic grafts of intact bone marrow in immunologically tolerant recipients and after abrogation of the tolerance.

In ossicles derived from grafts of compatible intact bone marrow in the subcapsular renal site the ectopic bone remains of donor provenance but the haemopoietic elements are partially replaced with tissue by host cells derived from stem cells migrating from the blood. Ossicles derived from incompatible intact bone marrow grafted in immunologically tolerant recipients are morphologically identical to those derived from compatible intact bone marrow. On abolition of the tolerance the allogeneic ectopic bone and donor microenvironment for haemopoiesis became manifest: haemopoietic marrow, even the host elements, was rapidly lost, bone disappearing rapidly if recently established, slowly if long established. Provided the state of immunological tolerance persists, the histoincompatible bone and microenvironment of the donor co-exist with marrow derived from the circulating haemopoietic stem cells of the tolerant recipient.     

The fate of donor osteocytes in fine particulate bone powders during repair of bone defects in experimental rats

The aim of the study was to investigate the fate of donor osteocytes in fine particulate bone powders during repair of bone defects in experimental rats. The iliac bone of male inbred DA rats was harvested and used as the larger bone grafts and also prepared as fine particulate (granulated) bone powders (300-500 μm size particles) for transplantation into radial defects in female rats. The presence and relative amounts of genes specific to the sex-determining region of the Y-chromosome (Sry) originating from the bone grafts were evaluated by polymerase chain reaction and by in situ hybridization, respectively. Additional samples were evaluated histologically. In the larger bone grafts, the expression of Sry decreased relatively early, disappeared by 1 week, reappeared at 4 weeks and continued to increase with time. In the fine particulate bone powders, Sry was detected all the time and its expression was statistically greater than in the larger bone grafts at each time point. Both bone grafts provided donor cells to repair the defects. The donor cells seemed to function differently between the two groups. The fine particulate bone powders contained more living osteocytes in comparison with the larger bone grafts and may accelerate the healing of bone defects compared with conventional autografts.     

Effect of spaceflight on periosteal bone formation in rats

Male Wistar rats were placed in orbit for 18.5 days aboard the Soviet COSMOS 1129 biological satellite. Tetracycline was administered before and after spaceflight to label areas of bone formation. An inhibition of periosteal bone formation occurred during spaceflight in the tibial and humeral diaphyses, but this defect was corrected during the postflight period. The increased extent of arrest lines at these skeletal sites suggested that periosteal bone formation may have even ceased during spaceflight. The rib exhibited a small but nonsignificant decrease in periosteal bone formation. Endosteal bone resorption was not affected markedly by spaceflight conditions. The observed inhibition of periosteal bone formation may be a result of mechanical unloading, but endocrine factors cannot be ruled out.     

Influence of the in vitro culture period on the in vivo performance of cell/titanium bone tissue-engineered constructs using a rat cranial critical size defect model

The aim of this study was to investigate the in vivo performance in bone-regenerating capability of cell/scaffold constructs implanted into an orthotopic site. Bone marrow stromal osteoblasts were seeded on titanium fiber mesh scaffolds using a cell suspension (5 x 10(5) cells per scaffold) and cultured for 1, 4, and 8 days under either static or flow perfusion conditions forming six different treatment groups. A total of 16 constructs from each one of the six treatment groups were then implanted into an 8-mm critical size calvarial defect created in the cranium of adult syngeneic male Fisher rats. Half of the constructs from each group were retrieved 7 days postimplantation, and the other half of the constructs were retrieved 30 days postimplantation and examined for new bone formation and tissue response. Constructs retrieved 7 days postimplantation were filled with fibrous tissue and capillaries, but no bone formation was observed in any of the six treatment groups. Constructs retrieved 30 days postimplantation showed bone formation (at least 7 out of 8 constructs in all treatment groups). Titanium fiber meshes seeded with bone marrow stromal osteoblasts and cultured for 1 day under flow perfusion conditions before implantation appeared to give the highest percentage of bone formation per implant (64 +/- 17%). They also showed the highest ratio of critical size cranial defects that resulted in union of the defect 30 days postimplantation (7 out of 8) together with the constructs cultured for 1 day under static conditions before implantation. There were no significant differences between the different treatment groups; this finding is most likely due to the large variability of the results and the small number of animals per group. However, these results show that titanium fiber mesh scaffolds loaded with bone marrow stromal osteoblasts can have osteoinductive properties when implanted in an orthotopic site. They also indicate the importance of the stage of the osteoblastic differentiation and the quality of the in vitro generated extracellular matrix in the observed osteoinductive potential.     

Periosteal donor site regeneration in rats

Periosteal regeneration was investigated in two periosteal donor sites of the femur. The periosteum was taken from the femur epiphyses and diaphyses of 32 rats. The animals were sacrificed 1, 2, 3, 4 and 8 weeks after periosteal stripping. Intense cell proliferation occurred in the first week. After two weeks, a thick tissue layer formed by osteoblasts and undifferentiated cells was seen at the two donor sites. Eight weeks after, the periosteum had the same aspect as that from the right femur, which was used as control. Histomorphometric analysis showed that periosteal regeneration was significantly different between epiphyses and diaphyses. Periosteal regeneration at donor site located in epiphyses presented greater proliferation and better osteogenic activity than that observed in diaphyses.     

The nature and role of periosteum in bone and cartilage regeneration

This study was undertaken to determine whether periosteum from different bone sources in a donor results in the same formation of bone and cartilage. In this case, periosteum obtained from the cranium and mandible (examples of tissue supporting intramembranous ossification) and the radius and ilium (examples of tissues supporting endochondral ossification) of individual calves was used to produce tissue-engineered constructs that were implanted in nude mice and then retrieved after 10 and 20 weeks. Specimens were compared in terms of their osteogenic and chondrogenic potential by radiography, histology, and gene expression levels. By 10 weeks of implantation and more so by 20 weeks, constructs with cranial periosteum had developed to the greatest extent, followed in order by ilium, radius, and mandible periosteum. All constructs, particularly with cranial tissue although minimally with mandibular periosteum, had mineralized by 10 weeks on radiography and stained for proteoglycans with safranin-O red (cranial tissue most intensely and mandibular tissue least intensely). Gene expression of type I collagen, type II collagen, runx2, and bone sialoprotein (BSP) was detectable on QRT-PCR for all specimens at 10 and 20 weeks. By 20 weeks, the relative gene levels were: type I collagen, ilium > radial ≥ cranial ≥ mandibular; type II collagen, radial > ilium > cranial ≥ mandibular; runx2, cranial > radial > mandibular ≥ ilium; and BSP, ilium ≥ radial > cranial > mandibular. These data demonstrate that the osteogenic and chondrogenic capacity of the various constructs is not identical and depends on the periosteal source regardless of intramembranous or endochondral ossification. Based on these results, cranial and mandibular periosteal tissues appear to enhance bone formation most and least prominently, respectively. The appropriate periosteal choice for bone and cartilage tissue engineering and regeneration should be a function of its immediate application as well as other factors besides growth rate.     

Bone morphogenetic protein-transduced human fibroblasts convert to osteoblasts and form bone in vivo

Experimental cell or ex vivo gene therapy for localized bone formation typically uses osteoprogenitor cells propagated from periosteum or bone marrow. Both require bone or marrow biopsies to obtain cells. We have demonstrated that implantation of gingival or dermal fibroblasts transduced with BMP ex vivo, using a recombinant adenovirus (AdCMVBMP) attached to porous biodegradable scaffolds, form bone in vivo. Here we show that BMP-7-transduced fibroblasts suspended in injectable thermoset hydrogels form complete ossicles on subcutaneous injection and repair segmental defects in rat femurs. Bone formation was preceded by an intermediate cartilage stage. To determine the fate of the implanted transduced cells, thermoset hydrogel suspensions of ex vivo BMP-7-transduced or nontransduced fibroblasts were placed in diffusion chambers and implanted to allow development in vivo without direct contact with host cells. Only the BMP-transduced fibroblasts formed bone within the diffusion chambers in vivo, revealing that BMP transduction induces osteoblastic conversion of these cells.     

A novel approach to regenerating periodontal tissue by grafting autologous cultured periosteum

In the field of oral and maxillofacial surgery, tissue-engineering techniques have been found useful in regenerating lost tissues. Periodontal disease causes severe destruction of periodontal tissue, including the alveolar bone. In this study we attempted to regenerate canine periodontal tissue defects by grafting autologous cultured membrane derived from the periosteum. Under appropriate culture conditions, periosteal cells produce enough extracellular matrix to form sheets. Periosteum specimens were peeled from the mandibular body of adult hybrid dogs and were cultured until cells formed membrane. ALP activity was measured to determine an optimal time for grafting. The cultured periosteum (CP) was grafted and sutured on a mechanically made Class III furcation defect in the 4th mandibular premolars. After 3 months, the samples were harvested and observed radiologically and histologically. In cases of CP, the bone defects were regenerated and filled with newly formed hard tissue, whereas in the controls the defects remained. These results show that our novel treatment is effective in regenerating alveolar bone for the treatment of periodontal disease.