A mass guide for the harvest of cortico-cancellous bone from the posterior iliac crest for mandibular reconstruction.

This study sought to find the mass of particulate cortico-cancellous bone graft required per 1 cm continuity defect of the mandible. Harvested bone was weighed, milled and maximally compressed in a syringe. The defect length (DL) was measured in centimetres, and the compressed bone volume (CBV) used was recorded. The wet bone mass (WBM) of bone required per centimetre of mandibular defect, and the mass of bone yielding 1cc of compressed bone was calculated. Results were analysed statistically to determine if clinically meaningful differences exist between male and female iliac crest. Forty three patient records were reviewed (28 female). Thirty patients had bilateral, and 13 patients had unilateral iliac crest harvest. Mean WBM used per centimetre of mandible defect was 6.9 g.WBM required to produce 1cc of CBV was 2.0g. For the bilateral harvest group the mean DL was 10.3 cm, the mean WBM was 66.7 g, and the mean CBV was 33.9cc. There was no significant difference in mean WBM between male (72.8 g) and female (62 g) patients. The mean CBV for males (39.7 g) was significantly higher than females (29.5 g). For patients who had unilateral harvest the mean DL was 7.7 cm, the mean WBM harvested was 59.1 g, and the mean CBV was 29.4cc. The mean wet bone mass of posterior iliac crest required to graft each centimetre of mandibular segmental defect is 6.9 grams. A unilateral posterior iliac crest harvest will yield on average 59.1 grams of bone whilst a bilateral posterior iliac crest harvest will yield on average 66.7 grams.

New technique for endoscopically-assisted particulate graft reconstruction of the mandible.

The reconstruction of mandibular defects using particulate grafts is a proven technique that restores the osseous anatomy effectively. Secondary osseous reconstruction can be accomplished with endoscopic assistance and reduced-access incisions if an intermediate spacer is placed during resection. Two patients required reconstruction after resection of mandibular ameloblastomas. We used a modified protocol that involved the implantation of a graft of particulate corticocancellous bone after removal of the spacer, and prepared the recipient site under endoscopic guidance with small extraoral incisions. The grafts healed uneventfully and matured into ossicles suitable for the placement of osseointegrated implants.

Redefining the induction of periodontal tissue regeneration in primates by the osteogenic proteins of the transforming growth factor-ß supergene family.

The molecular bases of periodontal tissue induction and regeneration are the osteogenic proteins of the transforming growth factor-ß (TGF-ß) supergene family. These morphogens act as soluble mediators for the induction of tissues morphogenesis sculpting the multicellular mineralized structures of the periodontal tissues with functionally oriented ligament fibers into newly formed cementum. Human TGF-ß3 (hTGF-ß3 ) in growth factor-reduced Matrigel® matrix induces cementogenesis when implanted in class II mandibular furcation defects surgically prepared in the non-human primate Chacma baboon, Papio ursinus. The newly formed periodontal ligament space is characterized by running fibers tightly attached to the cementoid surface penetrating as mineralized constructs within the newly formed cementum assembling and initiating within the mineralized dentine. Angiogenesis heralds the newly formed periodontal ligament space, and newly sprouting capillaries are lined by cellular elements with condensed chromatin interpreted as angioblasts responsible for the rapid and sustained induction of angiogenesis. The inductive activity of hTGF-ß3 in Matrigel® matrix is enhanced by the addition of autogenous morcellated fragments of the rectus abdominis muscle potentially providing myoblastic, pericytic/perivascular stem cells for continuous tissue induction and morphogenesis. The striated rectus abdominis muscle is endowed with stem cell niches in para/perivascular location, which can be dominant, thus imposing stem cell features or stemness to the surrounding cells. This capacity to impose stemness is morphologically shown by greater alveolar bone induction and cementogenesis when hTGF-ß3 in Matrigel® matrix is combined with morcellated fragments of autogenous rectus abdominis muscle. The induction of periodontal tissue morphogenesis develops as a mosaic structure in which the osteogenic proteins of the TGF-ß supergene family singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis. In primates, the presence of several homologous yet molecularly different isoforms with osteogenic activity highlights the biological significance of this apparent redundancy and indicates multiple interactions during embryonic development and bone regeneration in postnatal life. Molecular redundancy with associated different biological functionalities in primate tissues may simply represent the fine-tuning of speciation-related molecular evolution in anthropoid apes at the early Pliocene boundary, which resulted in finer tuning of the bone induction cascade.

Tissue segregation restores the induction of bone formation by the mammalian transforming growth factor-ß(3) in calvarial defects of the non-human primate Papio ursinus.

A diffusion molecular hypothesis from the dura and/or the leptomeninges below that would control the induction of calvarial membranous bone formation by the recombinant human transforming growth factor-ß3 (hTGF-ß3) was investigated. Coral-derived calcium carbonate-based macroporous constructs (25 mm diameter; 3.5/4 mm thickness) with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) were inserted into forty calvarial defects created in 10 adult Chacma baboons Papio ursinus. In 20 defects, an impermeable nylon foil membrane (SupraFOIL(®)) was inserted between the cut endocranial bone and the underlying dura mater. Twenty of the macroporous constructs were preloaded with hTGF-ß3 (125 µg in 1000 µl 20 mM sodium succinate, 4% mannitol pH4.0), 10 of which were implanted into defects segregated by the SupraFOIL(®) membrane, and 10 into non-segregated defects. Tissues were harvested on day 90, processed for decalcified and undecalcified histology and quantitative real-time polymerase chain reaction (qRT-PCR). Segregated untreated macroporous specimens showed a reduction of bone formation across the macroporous spaces compared to non-segregated constructs. qRT-PCR of segregated untreated specimens showed down regulation of osteogenic protein-1 (OP-1), osteocalcin (OC), bone morphogenetic protein-2 (BMP-2), RUNX-2 and inhibitor of DNA binding-2 and -3 (ID2,ID3) and up regulation of TGF-ß3, a molecular signalling pathway inhibiting the induction of membranous bone formation. Non-segregated hTGF-ß3/treated constructs also showed non-osteogenic expression profiles when compared to non-segregated untreated specimens. Segregated hTGF-ß3/treated 7% HA/CC constructs showed significantly greater induction of bone formation across the macroporous spaces and, compared to non-segregated hTGF-ß3/treated constructs, showed up regulation of OP-1, OC, BMP-2, RUNX-2, ID2 and ID3. Similar up-regulated expression profiles were seen for untreated non-segregated constructs. TGF-ß signalling via ID genes creates permissive or refractory micro-environments that regulate the induction of calvarial bone formation which is controlled by the exogenous hTGF-ß3 upon segregation of the calvarial defects. The dura is the common regulator of the induction of calvarial bone formation modulated by the presence or absence of the SupraFOIL(®) membrane with or without hTGF-ß3.

Biological aspects of periodontal tissue regeneration: cementogenesis and the induction of Sharpey's fibres.

The realm of periodontal tissue regeneration has a plethora of unanswered questions and challenges due to the complexity of restoring mineralised and fibrous connective tissues ultimately to be covered by epithelium in a very specific spatial organisation. Wound healing of the periodontium follows a highly ordered sequence of events that guides cellular morphology, differentiation, migration and proliferation and comprises a series of cellular, extracellular and molecular reciprocal interactions. The prerequisite for regeneration of any structure is the trio of a soluble molecular signal, a scaffold and responding stem cells. Striated muscle represents an abundant source of easily accessible tissue that contains several perivascular, pericytic and myoblastic cell niches capable of differentiating and inducing selected tissue phenotypes and morphogenesis. Morcellated autogenous rectus abdominis muscle combined with 75 microg of hTGF-beta3 in Matrigel matrix implanted into non-human primate class II and III furcation defects induced greater alveolar bone formation and cementogenesis when compared to furcation defects without the addition of morcellated autogenous bone. In situ hybr disation and immunohistochemistry during embryonic development and tooth morphogenesis have shown synchronous but spatially different bone morphogenetic proteins (BMPs) expression during tissue induction and morphogenesis. Preclin cal studies in the non-human primate Papio ursinus have shown a seemingly specific cementogenic function of osteogenic protein-1 (OP-1, also known as BMP-7) when treating Class II furcation defects of Papio ursinus. In context, hOP-1 is preferentially cementogenic when implanted into non-human primate class II and III furcation defects whilst hBMP-2 is highly osteogenic but not cementogenic when in contact with dentine extracellular matrix. Importantly, naturally-derived highly purified BMPs/OPs, recombinant hOP-1 and hTGF-beta3 when implanted into non-human primates Class II and III furcation defects induce cementogenesis with morphologically and functionally oriented periodontal ligament fibres coursing within a newly formed highly vascular periodontal ligament space with Sharpey's fibres generated within the newly secreted cementoid matrix. The grand challenge of molecular and therapeutic implications is the biological significance of apparent redundancy. The presence of several homologous but molecularly different isoforms all endowed with the striking capacity of inducing "bone formation by autoinduction" indicates that there is a structure/activity profile amongst soluble osteogenic molecular signals; this suggests a therapeutic significance in clinical contexts. The structure/activity profile finely tunes the vast pleiotropic activities of the soluble molecular signals in mineralised and non-mineralised tissues profoundly modulating epithelial/mesenchymal tissue interactions. Significant advances in regenerative tissue engineering may be expected if ongoing research is tailored to provide further mechanistic and morphological insights into the relevance of the apparent redundancy and the structure/activity profile of the recombinant human osteogenic proteins. The presence of the structure/activity profile together with the biological significance of apparent redundancy will necessitate re-shaping and re-engineering developing newly devised targeted therapeutics for periodontal tissue engineering.

Synergistic induction of periodontal tissue regeneration by binary application of human osteogenic protein-1 and human transforming growth factor-ß3 in Class II furcation defects of Papio ursinus.

BACKGROUND AND OBJECTIVE: Binary applications of recombinant human osteogenic protein-1 (hOP-1) and transforming growth factor-ß3 (hTGF-ß3) synergize to induce pronounced bone formation. To induce periodontal tissue regeneration, binary applications of hOP-1 and hTGF-ß(3) were implanted in Class II furcation defects of the Chacma baboon, Papio ursinus. MATERIAL AND METHODS: Defects were created bilaterally in the furcation of the first and second mandibular molars of three adult baboons. Single applications of 25 µg hOP-1 and 75 µg hTGF-ß(3) in Matrigel(®) matrix were compared with 20:1 binary applications, i.e. 25 µg hOP-1 and 1.25 µg hTGF-ß(3). Morcellated fragments of autogenous rectus abdominis striated muscle were added to binary applications. Sixty days after implantation, the animals were killed and the operated tissues harvested en bloc. Undecalcified sections were studied by light microscopy, and regenerated tissue was assessed by measuring volume and height of newly formed alveolar bone and cementum. RESULTS: The hOP-1 and hTGF-ß(3) induced periodontal tissue regeneration and cementogenesis. Qualitative morphological analysis of binary applications showed clear evidence for considerable periodontal tissue regeneration. Quantitatively, the differences in the histomorphometric values did not reach statistical significance for the group size chosen for this primate study. The addition of morcellated muscle fragments did not enhance tissue regeneration. Binary applications showed rapid expansion of the newly formed bone against the root surfaces following fibrovascular tissue induction in the centre of the treated defects. CONCLUSION: Binary applications of hOP-1 and hTGF-ß(3) in Matrigel(®) matrix in Class II furcation defects of P. ursinus induced substantial periodontal tissue regeneration, which was tempered, however, by the anatomy of the furcation defect model, which does not allow for the rapid growth and expansion of the synergistic induction of bone formation, particularly when additionally treated with responding myoblastic stem cells.

Induction of bone formation by transforming growth factor-beta2 in the non-human primate Papio ursinus and its modulation by skeletal muscle responding stem cells.

OBJECTIVES: Four adult non-human primates Papio ursinus were used to study induction of bone formation by recombinant human transforming growth factor-beta(2) (hTGF-beta(2)) together with muscle-derived stem cells. MATERIALS AND METHODS: The hTGF-beta(2) was implanted in rectus abdominis muscles and in calvarial defects with and without addition of morcellized fragments of striated muscle, harvested from the rectus abdominis or temporalis muscles. Expression of osteogenic markers including osteogenic protein-1, bone morphogenetic protein-3 and type IV collagen mRNAs from generated specimens was examined by Northern blot analysis. RESULTS: Heterotopic intramuscular implantation of 5 and 25 microg hTGF-beta(2) combined with 100 mg of insoluble collagenous bone matrix yielded large corticalized mineralized ossicles by day 30 with remodelling and induction of haematopoietic marrow by day 90. Addition of morcellized rectus abdominis muscle to calvarial implants enhanced induction of bone formation significantly by day 90. CONCLUSIONS: In Papio ursinus, in marked contrast to rodents and lagomorphs, hTGF-beta(2) induced large corticalized and vascularized ossicles by day 30 after implantation into the rectus abdominis muscle. This striated muscle contains responding stem cells that enhance the bone induction cascade of hTGF-beta(2). Induction of bone formation by hTGF-beta(2) in the non-human primate Papio ursinus may occur as a result of expression of bone morphogenetic proteins on heterotopic implantation of hTGF-beta(2); the bone induction cascade initiated by mammalian TGF-beta proteins in Papio ursinus needs to be re-evaluated for novel molecular therapeutics for induction of bone formation in clinical contexts.

Induction of cementogenesis and periodontal ligament regeneration by recombinant human transforming growth factor-beta3 in Matrigel with rectus abdominis responding cells.

BACKGROUND AND OBJECTIVE: In primates and in primates only, the transforming growth factor-b proteins induce endochondral bone formation. Transforming growth factor-b3 also induces periodontal tissue regeneration. Two regenerative treatments using human recombinant transforming growth factor-b3 were examined after implantation in mandibular furcation defects of the nonhuman primate, Papio ursinus. MATERIAL AND METHODS: Class III furcation defects were surgically created bilaterally in the mandibular first and second molars of two adult Chacma baboons (P. ursinus). Different doses of recombinant transforming growth factor-beta3 reconstituted with Matrigel matrix were implanted in the rectus abdominis muscle to induce heterotopic ossicles for subsequent transplantation to selected furcation defects. Twenty days after heterotopic implantation, periodontal defects were re-exposed, further debrided and implanted with minced fragments of induced heterotopic ossicles. Contralateral class III furcation defects were implanted directly with recombinant transforming growth factor-beta3 in Matrigel matrix with the addition of minced fragments of autogenous rectus abdominis muscle. Treated quadrants were not subjected to oral hygiene procedures so as to study the effect of the direct application of the recombinant morphogen in Matrigel on periodontal healing. Histomorphometric analyses on undecalcified sections cut from specimen blocks harvested on day 60 measured the area of newly formed alveolar bone and the coronal extension of the newly formed cementum along the exposed root surfaces. RESULTS: Morphometric analyses showed greater alveolar bone regeneration and cementogenesis in furcation defects implanted directly with 75 microg of transforming growth factor-beta3 in Matrigel matrix with the addition of minced muscle tissue. CONCLUSION: Matrigel matrix is an optimal delivery system for the osteogenic proteins of the transforming growth factor-beta superfamily, including the mammalian transforming growth factor-beta3 isoform. The addition of minced fragments of rectus abdominis muscle provides responding stem cells for further tissue induction and morphogenesis by the transforming growth factor-beta3 protein.

Cementogenesis and the induction of periodontal tissue regeneration by the osteogenic proteins of the transforming growth factor-beta superfamily.

The antiquity and severity of periodontal diseases are demonstrated by the hard evidence of alveolar bone loss in gnathic remains of the Pliocene/Pleistocene deposits of the Bloubank Valley at Sterkfontein, Swartkrans and Kromdrai in South Africa. Extant Homo has characterized and cloned a superfamily of proteins which include the bone morphogenetic proteins that regulate tooth morphogenesis at different stages of development as temporally and spatially connected events. The induction of cementogenesis, periodontal ligament and alveolar bone regeneration are regulated by the co-ordinated expression of bone morphogenetic proteins. Naturally derived and recombinant human bone morphogenetic proteins induce periodontal tissue regeneration in mammals. Morphological analyses on undecalcified sections cut at 3-6 mum on a series of mandibular molar Class II and III furcation defects induced in the non-human primate Papio ursinus show the induction of cementogenesis. Sharpey's fibers nucleate as a series of composite collagen bundles within the cementoid matrix in close relation to embedded cementocytes. Osteogenic protein-1 and bone morphogenetic protein-2 possess a structure-activity profile, as shown by the morphology of tissue regeneration, preferentially cementogenic and osteogenic, respectively. In Papio ursinus, transforming growth factor-beta(3) also induces cementogenesis, with Sharpey's fibers inserting into newly formed alveolar bone. Capillary sprouting and invasion determine the sequential insertion and alignment of individual collagenic bundles. The addition of responding stem cells prepared by finely mincing fragments of autogenous rectus abdominis muscle significantly enhances the induction of periodontal tissue regeneration when combined with transforming growth factor-beta(3) implanted in Class II and III furcation defects of Papio ursinus.

The induction of bone formation by smart biphasic hydroxyapatite tricalcium phosphate biomimetic matrices in the non-human primate Papio ursinus.

Long-term studies in the non-human primate Chacma baboon Papio ursinus were set to investigate the induction of bone formation by biphasic hydroxyapatite/p-tricalcium phosphate (HA/beta-TCP) biomimetic matrices. HA/beta-TCP biomimetic matrices in a pre-sinter ratio (wt%) of 40/60 and 20/80, respectively, were sintered and implanted in the rectus abdominis and in calvarial defects of four adult baboons. The post-sinter phase content ratios were 19/81 and 4/96, respectively. Morphological analyses on day 90 and 365 showed significant induction of bone formation within concavities of the biomimetic matrices with substantial bone formation by induction and resorption/dissolution of the implanted matrices. One year after implantation in calvarial defects, 4/96 biphasic biomimetic constructs showed prominent induction of bone formation with significant dissolution of the implanted scaffolds. The implanted smart biomimetic matrices induce de novo bone formation even in the absence of exogenously applied osteogenic proteins of the transforming growth factor-beta(TGF-beta) superfamily. The induction of bone formation biomimetizes the remodelling cycle of the cortico-cancellous bone of primates whereby resorption lacunae, pits and concavities cut by osteoclastogenesis are regulators of bone formation by induction. The concavities assembled in HA/beta-TCP biomimetic bioceramics are endowed with multifunctional pleiotropic self-assembly capacities initiating and promoting angiogenesis and bone formation by induction. Resident mesenchymal cells differentiate into osteoblastic cell lines expressing, secreting and embedding osteogenic soluble molecular signals of the TGF-beta superfamily within the concavities of the biomimetic matrices initiating bone formation as a secondary response.

Periodontal tissue regeneration by recombinant human transforming growth factor-beta 3 in Papio ursinus.

BACKGROUND AND OBJECTIVE: Osteogenic proteins of the transforming growth factor-beta superfamily induce periodontal tissue regeneration in animal models, including primates. To our knowledge, no studies have been performed in periodontal regeneration using the transforming growth factor-beta 3 isoform. In the present study, recombinant human transforming growth factor-beta 3 was examined for its ability to induce periodontal tissue regeneration in the nonhuman primate, Papio ursinus. MATERIAL AND METHODS: Class II furcation defects were surgically created bilaterally in the maxillary and mandibular molars of four adult baboons. Heterotopic ossicles, for transplantation to selected furcation defects, were induced within the rectus abdominis muscle by recombinant human transforming growth factor-beta 3. Forty days later, the periodontal defects were implanted with recombinant human transforming growth factor-beta 3 in Matrigel as the delivery system, with recombinant human transforming growth factor-beta 3 plus minced muscle tissue in Matrigel, or with the harvested recombinant human transforming growth factor-beta 3-induced ossicles. Sixty days after periodontal implantation, the animals were killed and the specimens harvested. Histological analysis on undecalcified sections measured the area and volume of new alveolar bone and the coronal extension of newly formed alveolar bone and cementum. RESULTS: Morphometric analyses showed pronounced periodontal regeneration in experimental defects compared with controls. Substantial regeneration was observed in defects implanted with fragments of heterotopically induced ossicles and with recombinant human transforming growth factor-beta 3 plus minced muscle tissue. CONCLUSION: Recombinant human transforming growth factor-beta 3 in Matrigel significantly enhanced periodontal tissue regeneration in the nonhuman primate, P. ursinus.

Bone loss in the ovariectomized baboon Papio ursinus: densitometry, histomorphometry and biochemistry.

To develop a non-human primate model of systemic bone loss after ovariectomy, 24 ovariectomized (OVX) and eight control (non-OVX) female baboons Papio ursinus were investigated over a period of 48 months using bone mineral density (BMD), iliac crest bone histomorphometry, bone turnover markers, and variables of calcium metabolism. Lumbar spine (L1-L4) BMD measured by dual energy X-ray absorptiometry (DXA) decreased in OVX animals in the first 12 months (-7.6%) and showed a slow trend towards recovery after 24 months. Controls showed a slow increase in spinal BMD over 4 years (+9.7%). Total hip BMD decreased slowly up to 48 months in all animals (OVX -12.6%versus controls -10%); this indicated that OVX had a limited effect on total hip BMD. Forearm BMD did not change. The significant decrease in trabecular bone volume (TBV) of the iliac crest from baseline to 12 months was followed by some recovery. Microarchitectural deterioration of trabecular bone in OVX animals was demonstrated by a decline in trabecular number and an increase in trabecular spacing. These changes were also evident on sections of whole vertebrae, proximal femora and iliac crests. Changes in iliac TBV reflected spinal but not hip BMD changes in the OVX animals. Static and dynamic histomorphometric variables indicated that bone turnover was increased for 36 months following OVX. Controls showed no changes in histomorphometric variables. Bone specific alkaline phosphatase (ALPs) in OVX animals remained elevated throughout the study; osteocalcin (OC) was significantly elevated only at 6 and 12 months, and deoxypyridinoline (Pyr-D) was elevated at 12 months but declined after 24 months. ALPs was thus more sensitive to the long-term effects of OVX than were OC or Pyr-D. Controls showed no changes in bone turnover markers. This study showed consistent deleterious changes in lumbar BMD, bone histomorphometry with microarchitectural deterioration together with altered biochemical markers of bone turnover in the first 12 months after OVX. Since these changes resemble those in post-menopausal women, the non-human primate Papio ursinus is suitable for the study of bone loss in post-menopausal women.

Recapitulating development: a template for periodontal tissue engineering.

The induction of bone formation by the soluble osteogenic molecular signals of the transforming growth factor-beta (TGF-beta) superfamily is a critical issue to periodontologists, molecular biologists, and tissue engineers alike, because preclinical studies in primates and clinical trials have demonstrated the bone induction capacity of bone morphogenetic and osteogenic proteins (BMPs/OPs) in clinical context. BMPs/OPs, pleiotropic members of the TGF-beta superfamily, induce de novo endochondral bone formation as a recapitulation of embryonic development and act as soluble signals for tissue morphogenesis sculpting the multicellular mineralized structures of the periodontal tissues with functionally oriented periodontal ligament fibers inserting into newly formed cementum. This paper reviews the induction of the complex tissue morphologies of the periodontal tissues in the nonhuman primate Papio ursinus with furcation defects treated with doses of naturally derived and recombinantly produced human BMPs/OPs. Periodontal tissue regeneration develops as a mosaic structure in which the OPs of the TGF-beta superfamily singly, synergistically, and synchronously initiate and maintain tissue induction and morphogenesis.

Transformation of a prefabricated hydroxyapatite/osteogenic protein-1 implant into a vascularised pedicled bone flap in the human chest.

We describe the intramuscular transformation of a hydroxyapatite/osteogenic protein-1 (HA/OP-1) composite implant, into a vascularised pedicled bone flap useful for reconstruction of a hemi-mandible. Extraskeletal induction of a bone flap for transplantation was achieved without the addition of harvested bone, bone marrow, or stem cells. Five months after apparent clinical success, an MRSA infection of the graft led to its failure. The background to ectopically induced bone flaps is introduced, with our experience in a human case presented. The results from this emerging biotechnology are discussed in the light of limited human clinical experience.

Bone induction by recombinant human osteogenic protein-1 (hOP-1, BMP-7) in the primate Papio ursinus with expression of mRNA of gene products of the TGF-beta superfamily.

Predictable bone induction in clinical contexts requires information on the expression and cross regulation of gene products of the transforming growth factor-beta (TGF-beta) superfamily elicited by single applications of each recombinant human bone morphogenetic/osteogenic proteins (BMPs/OPs). Using the calvarium and the rectus abdominis muscle of adult baboons Papio ursinus as a model for tissue induction and morphogenesis, this study investigated the induction of bone morphogenesis by gamma-irradiated hOP-1 delivered by gamma-irradiated bovine insoluble collagenous bone matrix, the hOP-1 osteogenic device, for bone induction in heterotopic and orthotopic sites of the primate Papio ursinus and the expression patterns of OP-1, collagen type IV, BMP-3 and TGFbeta1mRNAs elicited by increasing single applications of doses of the hOP-1 osteogenic devices (0.1, 0.5 and 2.5 mg hOP-1/g of matrix) applied heterotopically in the rectus abdominis muscle and orthotopically in 48 calvarial defects of 12 adult baboons. Histology and histomorphometry on serial undecalcified sections prepared from the specimens harvested on day 15, 30 and 90 showed that all the doses of the hOP-1 osteogenic device induced bone formation culminating in complete calvarial regeneration by day 90. Type IV collagen mRNA expression, a marker of angiogenesis, was strongly expressed in both heterotopic and orthotopic tissues. High levels of expression of OP-1 mRNA demonstrated autoinduction of OP-1 mRNAs. Expression levels of BMP-3 mRNA varied from tissues induced in heterotopic vs. orthotopic sites with high expression in rapidly forming heterotopic ossicles together with high expression of type IV collagen mRNA. The temporal and spatial expressions of TGF-beta1 mRNAindicate a specific temporal transcriptional window during which expression of TGF-beta1 is mandatory for successful and optimal osteogenesis. The induction of bone by hOP-1 in Papio ursinus develops as a mosaic structure with distinct spatial and temporal patterns of gene expression of members of the TGF-beta superfamily that singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis.

Soluble, insoluble and geometric signals sculpt the architecture of mineralized tissues.

Bone morphogenetic and osteogenic proteins (BMPs/OPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are soluble mediators of tissue morphogenesis and induce de novo endochondral bone formation in heterotopic extraskeletal sites as a recapitulation of embryonic development. In the primate Papio ursinus, the induction of bone formation has been extended to the TGF-beta isoforms per se. In the primate and in the primate only, the TGF-beta isoforms are initiators of endochondral bone formation by induction and act in a species-, site- and tissue-specific mode with robust endochondral bone induction in heterotopic sites but with limited new bone formation in orthotopic bone defects. The limited inductive capacity orthotopically of TGF-beta isoforms is associated with expression of the inhibitory Smads, Smad6 and Smad7. In primates, bone formation can also be induced using biomimetic crystalline hydroxyapatite matrices with a specific surface geometry and without the exogenous application of osteogenic proteins of the TGF-beta superfamily, even when the biomimetic matrices are implanted heterotopically in the rectus abdominis muscle. The sequence of events that directs new bone formation upon the implantation of highly crystalline biomimetic matrices initiates with vascular invasion, mesenchymal cell migration, attachment and differentiation of osteoblast-like cells attached to the substratum, expression and synthesis of osteogenic proteins of the TGF-beta superfamily resulting in the induction of bone as a secondary response. The above findings in the primate indicate enormous potential for the bioengineering industry. Of particular interest is that biomimetic matrices with intrinsic osteoinductivity would be an affordable option in the local context.

Periodontal tissue regeneration by combined applications of recombinant human osteogenic protein-1 and bone morphogenetic protein-2. A pilot study in Chacma baboons (Papio ursinus).

Native and recombinant human bone morphogenetic/osteogenic proteins (BMPs/ OPs) singly initiate bone induction in vivo. The finding of synchronous but spatially different BMPs/OPs expression during periodontal tissue morphogenesis suggests novel therapeutic approaches using morphogen combinations based on recapitulation of embryonic development. Twelve furcation defects prepared in the first and second mandibular molars of three adult baboons (Papio ursinus) were used to assess whether qualitative histological aspects of periodontal tissue regeneration could be enhanced and tissue morphogenesis modified by combined or single applications of recombinant hOP-1 and hBMP-2. Doses of BMPs/OPs were 100 microg of each protein per 1 g of insoluble collagenous bone matrix as carrier. Approximately 200 mg of carrier matrix was used per furcation defect. Undecalcified sections cut for histological analysis 60 d after healing of hOP-1-treated specimens showed substantial cementogenesis with scattered remnants of the collagenous carrier. hBMP-2 applied alone induced greater amounts of mineralized bone and osteoid when compared to hOP-1 alone or to combined morphogen applications. Combined applications of hOP-1 and hBMP-2 did not enhance alveolar bone regeneration or new attachment formation over and above the single applications of the morphogens. The results of this study, which is the first to attempt to address the structure-activity relationship amongst BMP/OP family members, indicate that tissue morphogenesis induced by hOP-1 and hBMP-2 is qualitatively different when the morphogens are applied singly, with hOP-1 inducing substantial cementogenesis. hBMP-2 treated defects, on the other hand, showed limited cementum formation but a temporal enhancement of alveolar bone regeneration and remodelling. The demonstration of therapeutic mosaicism in periodontal regeneration will require extensive testing of ratios and doses of recombinant morphogen combinations for optimal tissue engineering in clinical contexts.

Bone induction by BMPs/OPs and related family members in primates.

BACKGROUND: In a series of studies in the primate Papio ursinus, we have examined the capacity of bone morphogenetic proteins (BMPs/OPs) delivered in a variety of biomaterial carrier systems to elicit bone formation in heterotopic and orthotopic sites. In this review, we compare the osteoinductive effects of different biomaterial delivery systems that have or have not been pretreated with BMPs/OPs. In particular, we focus on the geometric induction of bone formation by sintered porous hydroxyapatite (SPHA) discs with concavities on their planar surfaces, which elicit bone formation without exogenously applied BMPs/OPs. METHODS: Heterotopic bone formation was examined by bilaterally implanting 100-mg pellets of a collagenous carrier containing BMPs/OPs in the rectus abdominis muscle of the adult baboon. Orthotopic bone formation was examined by implanting 1 g of a collagenous carrier containing BMPs/OPs into two full-thickness critical-sized 25-mm-diameter defects on each side of the calvaria of adult baboons. The BMPs/OPs whose effects were examined included recombinant human osteogenic protein-1 (rhOP-1), recombinant human transforming growth factor-beta1 (rhTGF-beta1), rhTGF-beta2, and porcine platelet derived transforming growth factor-beta1 (pTGF-beta1). Tissue from the rectus abdominis muscle was harvested 30 or 90 days after implantation. Tissue from the orthotopic calvarial model was examined at 1, 3, 6, 9, and 12 months after implantation. To demonstrate the effect of surface geometry on bone induction, hydroxyapatite powders were sintered to form solid discs with a series of concavities on the planar surfaces of the SPHA discs. The discs were either pretreated with exogenous rhOP-1 or not treated with exogenous OP-1. They were then implanted heterotopically or orthotopically into calvarial defects. Bone formation was evaluated histologically in undecalcified sections stained with Goldner's trichrome stain or 0.1% toluidine blue. RESULTS: Naturally derived BMPs/OPs or rhOP-1 in a collagenous carrier elicit heterotopic bone formation and the complete healing of 25-mm-diameter critical-sized defects by day 90 following implantation. Binary applications of TGF-beta1 together with rhOP-1 in the collagen carrier induced massive endochondral ossicles in heterotopic sites and bone formation in calvarial defects. pTGF-beta1, rhTGF-beta1, and rhTGF-beta2 are powerful inducers of heterotopic endochondral bone formation but elicit limited bone formation in calvarial defects. SPHA discs pretreated with rhOP-1 elicited extensive bone formation in both heterotopic and orthotopic sites. However, SPHA without rhOP-1 also elicited bone formation in heterotopic and orthotopic sites and complete healing of the calvarial defects. CONCLUSION: We have prepared SPHA discs with concavities on their planar surfaces that induce bone formation in heterotopic or orthotopic critical-sized calvarial defects without exogenously applied BMPs/OPs. This biomaterial induces bone formation by intrinsic osteoinductivity regulated by the geometry of the substratum. The incorporation of specific biological activities into biomaterials by manipulating the geometry of the substratum, defined as geometric induction of bone formation, may make it possible to engineer morphogenetic responses for therapeutic osteogenesis in clinical contexts. CLINICAL RELEVANCE: We have implemented a clinical trial using naturally derived BMPs/OPs extracted and purified from bovine bone matrices and implanted in craniofacial defects in humans. In addition, the discovery that specific geometric and surface characteristics of sintered hydroxyapatites can induce intrinsic osteoinductivity in primates paves the way for formulation and therapeutic application of porous substrata designed to obtain predictable intrinsic osteoinductivity in clinical contexts.

Induction of bone formation by recombinant human osteogenic protein-1 and sintered porous hydroxyapatite in adult primates.

A critical issue in tissue engineering and morphogenesis of bone is the development of novel biomimetic biomaterials that are capable of optimizing the biological activity of recombinant human bone morphogenetic and osteogenic proteins, which are molecules that initiate bone formation in vivo. From a therapeutic perspective, a carrier matrix is required for the local delivery of these proteins to evoke a desired osteogenic effect. In view of the affinity of these proteins for hydroxyapatite, which may reflect the in vivo supramolecular assembly of bone proteins bound to both the extracellular matrix and the mineral component of bone, we investigated the efficacy of single applications of different doses of human osteogenic protein-1 (hOP-1) adsorbed onto sintered porous hydroxyapatites for bone induction in orthotopic calvarial defects in 12 adult male baboons (Papio ursinus) and heterotopically in the rectus abdominis of four additional baboons. In orthotopic specimens, pretreatment of sintered porous hydroxyapatites with 100 microgram of hOP-1 in 500 microliter of 5 mM hydrochloric acid resulted in rapid and diffuse osteoinduction restricted within the porous spaces of the hydroxyapatite, as evaluated by histology and histomorphometry on day 30. Hydroxyapatites treated with 500 microgram of hOP-1 showed a different pattern of bone formation and distribution on day 30 as compared with the lower dose of the recombinant morphogen. Although bone formation was extensive with the higher dose, it was found on the endocranial and pericranial aspects of the specimens, enveloping the implanted hydroxyapatite carrier, and the internal porous spaces were occupied by a rich vascular network without any bone formation. By 90 and 365 days after the implantation of both doses of hOP-1, however, there was remodelling and complete penetration of the newly induced bone within the available porous spaces. The combination of hOP-1 and hydroxyapatite also showed extensive bone formation in heterotopic specimens harvested from the rectus abdominis muscle of the baboon using doses of 5, 25, and 45 microgram of hOP-1 per implant. These findings in the adult primate demonstrate extensive bone formation by hOP-1 adsorbed onto sintered porous hydroxyapatites and suggest that predictable osteogenesis in clinical contexts for treatment of craniofacial bone defects may be engineered using inorganic, nonimmunogenic, and carvable delivery systems that initiate osteogenesis with relatively low doses of recombinant osteogenic proteins, thus mimicking the macrostructure and microstructure of living bone.

Transforming growth factor-beta1 supports the rapid morphogenesis of heterotopic endochondral bone initiated by human osteogenic protein-1 via the synergistic upregulation of molecular markers.

Members of the transforming growth factor-beta (TGF-beta) superfamily of proteins, the bone morphogenetic proteins (BMPs) and the TGF-beta isoforms, are involved in the coordination of cartilage and bone differentiation both in embryonic development and in postnatal life. Both osteogenic protein-1 (OP-1) and TGF-beta1 have been shown to be potent regulators and inducers of heterotopic endochondral bone induction in non-human primates. In marked contrast, TGF-beta1 does not induce heterotopic endochondral bone in rodents. In the primate, the osteogenic properties of OP-1 are synergistically enhanced by the combined administration of TGF-beta1. The binary application of OP-1 (0.1, 0.3, 1.0 and 3.0 microg) and TGF-beta1 (0.01, 0.03 and 0.1 microg) to 25 mg of guanidinium-inactivated insoluble collagenous bone matrix as carrier in the rodent heterotopic bioassay for 7, 12 and 21 days resulted in a classical synergistic, dose-dependent and temporal up-regulation of OP-1-induced endochondral bone formation. There were significant increases in alkaline phosphatase activity (day 12) and calcium content (days 12 and 21). mRNA expression of OP-1, TGF-beta1, BMP-3 and collagens type II and IV, markers of bone formation, showed an up-regulation of the genes (days 12 and 21) by the binary applications of the morphogens. Histologically, single applications of OP-1 elicited a dose dependent induction of endochondral bone formation while the binary applications resulted in a temporal acceleration of the morphogenetic cascade. The optimal ratio of OP-1/TGF-beta1 was 30:1 by weight for endochondral bone formation and expression of molecular markers. The present data provides insights to the mechanisms of synergistic molecular therapeutics for endochondral bone formation in clinical contexts.