Trauma induced tissue survival in vitro with a muscle-biomaterial based osteogenic organoid system: a proof of concept study.

BACKGROUND: The translation from animal research into the clinical environment remains problematic, as animal systems do not adequately replicate the human in vivo environment. Bioreactors have emerged as a good alternative that can reproduce part of the human in vivo processes at an in vitro level. However, in vitro bone formation platforms primarily utilize stem cells only, with tissue based in vitro systems remaining poorly investigated. As such, the present pilot study explored the tissue behavior and cell survival capability within a new in vitro skeletal muscle tissue-based biomaterial organoid bioreactor system to maximize future bone tissue engineering prospects. RESULTS: Three dimensional printed ß-tricalcium phosphate/hydroxyapatite devices were either wrapped in a sheet of rat muscle tissue or first implanted in a heterotopic muscle pouch that was then excised and cultured in vitro for up to 30 days. Devices wrapped in muscle tissue showed cell death by day 15. Contrarily, devices in muscle pouches showed angiogenic and limited osteogenic gene expression tendencies with consistent TGF-ß1, COL4A1, VEGF-A, RUNX-2, and BMP-2 up-regulation, respectively. Histologically, muscle tissue degradation and fibrin release was seen being absorbed by devices acting possibly as a support for new tissue formation in the bioceramic scaffold that supports progenitor stem cell osteogenic differentiation. CONCLUSIONS: These results therefore demonstrate that the skeletal muscle pouch-based biomaterial culturing system can support tissue survival over a prolonged culture period and represents a novel organoid tissue model that with further adjustments could generate bone tissue for direct clinical transplantations.

Synergistic interaction of hTGF-ß3 with hBMP-6 promotes articular cartilage formation in chitosan scaffolds with hADSCs: implications for regenerative medicine.

BACKGROUND: Human TGF-ß3 has been used in many studies to induce genes coding for typical cartilage matrix components and accelerate chondrogenic differentiation, making it the standard constituent in most cultivation media used for the assessment of chondrogenesis associated with various stem cell types on carrier matrices. However, in vivo data suggests that TGF-ß3 and its other isoforms also induce endochondral and intramembranous osteogenesis in non-primate species to other mammals. Based on previously demonstrated improved articular cartilage induction by a using hTGF-ß3 and hBMP-6 together on hADSC cultures and the interaction of TGF- ß with matrix in vivo, the present study investigates the interaction of a chitosan scaffold as polyanionic polysaccharide with both growth factors. The study analyzes the difference between chondrogenic differentiation that leads to stable hyaline cartilage and the endochondral ossification route that ends in hypertrophy by extending the usual panel of investigated gene expression and stringent employment of quantitative PCR. RESULTS: By assessing the viability, proliferation, matrix formation and gene expression patterns it is shown that hTGF-ß3 + hBMP-6 promotes improved hyaline articular cartilage formation in a chitosan scaffold in which ACAN with Col2A1 and not Col1A1 nor Col10A1 where highly expressed both at a transcriptional and translational level. Inversely, hTGF-ß3 alone tended towards endochondral bone formation showing according protein and gene expression patterns. CONCLUSION: These findings demonstrate that clinical therapies should consider using hTGF-ß3 + hBMP-6 in articular cartilage regeneration therapies as the synergistic interaction of these morphogens seems to ensure and maintain proper hyaline articular cartilage matrix formation counteracting degeneration to fibrous tissue or ossification. These effects are produced by interaction of the growth factors with the polysaccharide matrix.

The effect of autologous Achilles bursal tissue implants in tendon-to-bone healing of rotator cuff tears in rats.

BACKGROUND: The aim of this study was to investigate the influence of autologous bursal tissue derived from the Achilles bursa on tendon-to-bone healing after rotator cuff tear repair in a rat model. METHODS: A total of 136 Sprague-Dawley rats were randomly assigned to either an untreated or a bursal tissue application group or biomechanical testing and histologic testing after rotator cuff repair. After separating the supraspinatus tendon close to the greater tuberosity, the tendon was reattached either unaltered or with a bursal tissue interposition sewn onto the interface. Immunohistologic analysis was performed 1 and 7 weeks after supraspinatus tendon reinsertion. Biomechanical testing of the tendon occurred 6 and 7 weeks after reinsertion. RESULTS: Immunohistologic results demonstrated a significantly higher percentage of Type II collagen (P = .04) after 1 and 7 weeks in the tendon-to-bone interface using autologous bursal tissue in comparison to control specimens. The bursa group showed a significantly higher collagen I to III quotient (P = .03) at 1 week after surgery in comparison to the 7-week postsurgery bursa groups and controls. Biomechanical assessment showed that overall tendon stiffness (P = .002) and the tendon viscoelasticity in the bursa group (P = .003) was significantly improved after 6 and 7 weeks. There was no significant difference (P = .55) in force to failure between the bursa group and the control group after 6 and 7 weeks. CONCLUSION: Autologous bursal tissue derived from the Achilles bursa and implanted to the tendon-to-bone interface after rotator cuff repair facilitates a faster healing response to re-establish the biologic and biomechanical integrity of the rotator cuff in rats.

Temporal TGF-ß Supergene Family Signalling Cues Modulating Tissue Morphogenesis: Chondrogenesis within a Muscle Tissue Model?

Temporal translational signalling cues modulate all forms of tissue morphogenesis. However, if the rules to obtain specific tissues rely upon specific ligands to be active or inactive, does this mean we can engineer any tissue from another? The present study focused on the temporal effect of "multiple" morphogen interactions on muscle tissue to figure out if chondrogenesis could be induced, opening up the way for new tissue models or therapies. Gene expression and histomorphometrical analysis of muscle tissue exposed to rat bone morphogenic protein 2 (rBMP-2), rat transforming growth factor beta 3 (rTGF-ß3), and/or rBMP-7, including different combinations applied briefly for 48 h or continuously for 30 days, revealed that a continuous rBMP-2 stimulation seems to be critical to initiate a chondrogenesis response that was limited to the first seven days of culture, but only in the absence of rBMP-7 and/or rTGF-ß3. After day 7, unknown modulatory effects retard rBMP-2s' effect where only through the paired-up addition of rBMP-7 and/or rTGF-ß3 a chondrogenesis-like reaction seemed to be maintained. This new tissue model, whilst still very crude in its design, is a world-first attempt to better understand how multiple morphogens affect tissue morphogenesis with time, with our goal being to one day predict the chronological order of what signals have to be applied, when, for how long, and with which other signals to induce and maintain a desired tissue morphogenesis.

Induction of Articular Chondrogenesis by Chitosan/Hyaluronic-Acid-Based Biomimetic Matrices Using Human Adipose-Derived Stem Cells.

Cartilage repair using tissue engineering is the most advanced clinical application in regenerative medicine, yet available solutions remain unsuccessful in reconstructing native cartilage in its proprietary form and function. Previous investigations have suggested that the combination of specific bioactive elements combined with a natural polymer could generate carrier matrices that enhance activities of seeded stem cells and possibly induce the desired matrix formation. The present study sought to clarify this by assessing whether a chitosan-hyaluronic-acid-based biomimetic matrix in conjunction with adipose-derived stem cells could support articular hyaline cartilage formation in relation to a standard chitosan-based construct. By assessing cellular development, matrix formation, and key gene/protein expressions during in vitro cultivation utilizing quantitative gene and immunofluorescent assays, results showed that chitosan with hyaluronic acid provides a suitable environment that supports stem cell differentiation towards cartilage matrix producing chondrocytes. However, on the molecular gene expression level, it has become apparent that, without combinations of morphogens, in the chondrogenic medium, hyaluronic acid with chitosan has a very limited capacity to stimulate and maintain stem cells in an articular chondrogenic state, suggesting that cocktails of various growth factors are one of the key features to regenerate articular cartilage, clinically.

Cementogenesis and osteogenesis in periodontal tissue regeneration by recombinant human transforming growth factor-ß3 : a pilot study in Papio ursinus.

OBJECTIVES: The aim of this study was to investigate cementogenesis and alveolar bone induction during in vivo periodontal tissue regeneration upon implantation of hTGF-ß3 in furcation defects of Papio ursinus and to evaluate the feasibility of gene expression studies. MATERIALS AND METHODS: Class II furcation defects (day 0) were prepared in mandibular first and second molars of three P. ursinus and on day 30 implanted with and without 75 µg hTGF-ß3 in Matrigel® matrix. On day 0, 30 and 90, cementum and alveolar bone were harvested for gene expression analyses. Coral-derived bioreactors with and without 250 µg hTGF-ß3 were implanted in the rectus abdominis to monitor tissue induction. RESULTS: hTGF-ß3 induced cementogenesis with TGF-ß3 , Cementum Protein-1 (Cemp1) and Osteocalcin (OC) up-regulation, and down-regulation of BMP-2 and OP-1. Matrigel® matrix specimens showed up-regulation of BMP-2, TGF-ß3 , and OC, with down-regulation of OP-1 and Cemp1. hTGF-ß3 induced alveolar bone with down-regulation of OP-1, TGF-ß3 , OC, and Cemp1. hTGF-ß3 bioreactors induced bone at the periphery only. BMP-3, BMP-4, TGF-ß1 and TGF-ß3 were up-regulated in the adjacent muscle with TGF-ß2 down-regulation. CONCLUSIONS: Cementogenesis and osteogenesis by hTGF-ß3 entail the expression and up-regulation of TGF-ß3 and OC with fine tuning and modulation of BMP-2 and OP-1.

The 3D-McMap Guidelines: Three-Dimensional Multicomposite Microsphere Adaptive Printing.

Microspheres, synthesized from diverse natural or synthetic polymers, are readily utilized in biomedical tissue engineering to improve the healing of various tissues. Their ability to encapsulate growth factors, therapeutics, and natural biomolecules, which can aid tissue regeneration, makes microspheres invaluable for future clinical therapies. While microsphere-supplemented scaffolds have been investigated, a pure microsphere scaffold with an optimized architecture has been challenging to create via 3D printing methods due to issues that prevent consistent deposition of microsphere-based materials and their ability to maintain the shape of the 3D-printed structure. Utilizing the extrusion printing process, we established a methodology that not only allows the creation of large microsphere scaffolds but also multicomposite matrices into which cells, growth factors, and therapeutics encapsulated in microspheres can be directly deposited during the printing process. Our 3D-McMap method provides some critical guidelines for issues with scaffold shape fidelity during and after printing. Carefully timed breaks, minuscule drying steps, and adjustments to extrusion parameters generated an evenly layered large microsphere scaffold that retained its internal architecture. Such scaffolds are superior to other microsphere-containing scaffolds, as they can release biomolecules in a highly controlled spatiotemporal manner. This capability permits us to study cell responses to the delivered signals to develop scaffolds that precisely modulate new tissue formation.

Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral-derived macroporous constructs.

Coral-derived calcium carbonate/hydroxyapatite macroporous constructs of the genus Goniopora with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) initiate the induction of bone formation. Which are the molecular signals that initiate pattern formation and the induction of bone formation? To evaluate the role of released calcium ions and osteoclastogenesis, 7% HA/CC was pre-loaded with either 500 µg of the calcium channel blocker, verapamil hydrochloride, or 240 µg of the osteoclast inhibitor, biphosphonate zoledronate, and implanted in the rectus abdominis muscle of six adult Chacma baboons Papio ursinus. Generated tissues on days 15, 60 and 90 were analysed by histomorphometry and qRT-PCR. On day 15, up-regulation of type IV collagen characterized all the implanted constructs correlating with vascular invasion. Zoledronate-treated specimens showed an important delay in tissue patterning and morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal, if any, bone formation by induction. 7% HA/CC pre-loaded with the Ca(++) channel blocker verapamil hydrochloride strongly inhibited the induction of bone formation. Down-regulation of bone morphogenetic protein-2 (BMP-2) together with up-regulation of Noggin genes correlated with limited bone formation in 7% HA/CC pre-loaded with either verapamil or zoledronate, indicating that the induction of bone formation by coral-derived macroporous constructs is via the BMPs pathway. The spontaneous induction of bone formation is initiated by a local peak of Ca(++) activating stem cell differentiation and the induction of bone formation.

Parallel Chondrogenesis and Osteogenesis Tissue Morphogenesis in Muscle Tissue via Combinations of TGF-ß Supergene Family Members.

OBJECTIVE: This study aimed to decipher the temporal and spatial signaling code for clinical cartilage and bone regeneration. We investigated the effects of continuous equal dosages of a single, dual, or triplicate growth factor combination of bone morphogenetic protein (BMP)-2, transforming growth factor (TGF)-ß3, and/or BMP-7 on muscle tissue over a culturing period. The hypothesis was that specific growth factor combinations at specific time points direct tissue transformation toward endochondral bone or cartilage formation. DESIGN: The harvested muscle tissues from F-344 adult male rats were cultured in 96-well plates maintained in a specific medium and cultured at specific conditions. And the multidimensional and multi-time point analyses were performed at both the genetic and protein levels. RESULTS: The results insinuate that the application of growth factor stimulates a chaotic tissue response that does not follow a chronological signaling cascade. Both osteogenic and chondrogenic genes showed upregulation after induction, a similar result was also observed in the semiquantitative analysis after immunohistochemical staining against different antigens. CONCLUSIONS: The study showed that multiple TGF-ß superfamily proteins applied to tissue stimulate developmental tissue processes that do not follow current tissue formation rules. The findings contribute to the understanding of the chronological order of signals and expression patterns needed to achieve chondrogenesis, articular chondrogenesis, or osteogenesis, which is crucial for the development of treatments that can regrow bone and articular cartilage clinically.

Osteochondrogenesis by TGF-ß3, BMP-2 and noggin growth factor combinations in an ex vivo muscle tissue model: Temporal function changes affecting tissue morphogenesis.

In the absence of clear molecular insight, the biological mechanism behind the use of growth factors applied in osteochondral regeneration is still unresolved. The present study aimed to resolve whether multiple growth factors applied to muscle tissue in vitro, such as TGF-ß3, BMP-2 and Noggin, can lead to appropriate tissue morphogenesis with a specific osteochondrogenic nature, thereby revealing the underlying molecular interaction mechanisms during the differentiation process. Interestingly, although the results showed the typical modulatory effect of BMP-2 and TGF-ß3 on the osteochondral process, and Noggin seemingly downregulated specific signals such as BMP-2 activity, we also discovered a synergistic effect between TGF-ß3 and Noggin that positively influenced tissue morphogenesis. Noggin was observed to upregulate BMP-2 and OCN at specific time windows of culture in the presence of TGF-ß3, suggesting a temporal time switch causing functional changes in the signaling protein. This implies that signals change their functions throughout the process of new tissue formation, which may depend on the presence or absence of specific singular or multiple signaling cues. If this is the case, the signaling cascade is far more intricate and complex than originally believed, warranting intensive future investigations so that regenerative therapies of a critical clinical nature can function properly.

Biomimetic matrices self-initiating the induction of bone formation.

The new strategy of tissue engineering, and regenerative medicine at large, is to construct biomimetic matrices to mimic nature's hierarchical structural assemblages and mechanisms of simplicity and elegance that are conserved throughout genera and species. There is a direct spatial and temporal relationship of morphologic and molecular events that emphasize the biomimetism of the remodeling cycles of the osteonic corticocancellous bone versus the "geometric induction of bone formation," that is, the induction of bone by "smart" concavities assembled in biomimetic matrices of macroporous calcium phosphate-based constructs. The basic multicellular unit of the corticocancellous bone excavates a trench across the bone surface, leaving in its wake a hemiosteon rather than an osteon, that is, a trench with cross-sectional geometric cues of concavities after cyclic episodes of osteoclastogenesis, eventually leading to osteogenesis. The concavities per se are geometric regulators of growth-inducing angiogenesis and osteogenesis as in the remodeling processes of the corticocancellous bone. The concavities act as a powerful geometric attractant for myoblastic/myoendothelial and/or endothelial/pericytic stem cells, which differentiate into bone-forming cells. The lacunae, pits, and concavities cut by osteoclastogenesis within the biomimetic matrices are the driving morphogenetic cues that induce bone formation in a continuum of sequential phases of resorption/dissolution and formation. To induce the cascade of bone differentiation, the soluble osteogenic molecular signals of the transforming growth factor ß supergene family must be reconstituted with an insoluble signal or substratum that triggers the bone differentiation cascade. By carving a series of repetitive concavities into solid and/or macroporous biomimetic matrices of highly crystalline hydroxyapatite or biphasic hydroxyapatite/ß-tricalcium phosphate, we were able to embed smart biologic functions within intelligent scaffolds for tissue engineering of bone. The concavities assembled in the bioceramic constructs biomimetize the remodeling cycle of the corticocancellous bone and are endowed with multifunctional pleiotropic self-assembly capacities, initiating angiogenesis and bone formation by induction without the exogenous applications of the osteogenic-soluble molecular signals of the transforming growth factor ß supergene family. The incorporation of specific biologic activities into biomimetic matrices by manipulating the geometry of the substratum, defined as geometric induction of bone formation, is now helping to engineer therapeutic osteogenesis in clinical contexts.

Rotator Cuff Repair With Autologous Tenocytes and Biodegradable Collagen Scaffold: A Histological and Biomechanical Study in Sheep.

BACKGROUND: Large rotator cuff tears still represent a challenging problem in orthopaedics. The use of tenocytes on biomaterials/scaffolds for the repair of large rotator cuff defects might be a promising approach in the field of tendon regeneration. HYPOTHESIS: Cultivated autologous tenocytes seeded on a collagen scaffold lead to enhanced histological and biomechanical results after rotator cuff repair in a sheep model as compared with unseeded scaffolds in an acute setting. STUDY DESIGN: Controlled laboratory study. METHODS: At the tendon-bone junction of the infraspinatus tendon of the right foreleg of 24 sheep, a 3.5 × 1.5-cm tendon defect was created. Sheep were randomly allocated to group 1, a defect; group 2, where an unseeded collagen scaffold was implanted; or group 3, which received the implantation of a collagen scaffold seeded with autologous tenocytes. Twelve weeks postoperatively, tendon regeneration was examined histologically and biomechanically. RESULTS: The histology of the neotendons of group 3 showed better fiber patterns, a higher production of proteoglycans, and an increased genesis of collagen III in contrast to groups 1 and 2. Immunostaining revealed less tissue dedifferentiation, a more structured cartilage layer, and homogeneous cartilage-bone transition in group 3 in comparison with groups 1 and 2. Biomechanically, the tensile strength of the reconstructed tendons in group 3 (mean load to failure, 2516 N; SD, 407.5 N) was approximately 84% that of the native tendons (mean load to failure, 2995 N; SD, 223.1 N) without statistical significance. A significant difference (P = .0095) was registered between group 1 (66.9% with a mean load to failure of 2004 N; SD, 273.8 N) and the native tendons, as well as between group 2 (69.7% with a mean load to failure of 2088 N; SD, 675.4 N) and the native tendons for mean ultimate tensile strength. In breaking stress, a significant difference (P = .0095) was seen between group 1 (mean breaking stress, 1335 N/mm2; SD, 182.7 N/mm2) and the native tendons, as well as between group 2 (breaking stress, 1392 N/mm2; SD, 450.2 N/mm2) and the native tendons (mean breaking stress, 1996 N/mm2; SD, 148.7 N/mm2). Again, there was no significant difference between group 3 (mean breaking stress, 1677 N/mm2; SD, 271.7 N/mm2) and the native tendons. CONCLUSION: Autologous tenocytes seeded on collagen scaffolds yield enhanced biomechanical results after tendon-bone reconstruction as compared with unseeded scaffolds in an acute setting. Biomechanical results and histological outcomes were promising, showing that the use of autologous tenocytes with specific carrier matrices could be a novel approach for repairing rotator cuff tears. CLINICAL RELEVANCE: This study supports the use of tenocytes and scaffolds for improving the quality of tendon-bone regeneration.

Gene activated adipose tissue fragments as advanced autologous biomaterials for bone regeneration: osteogenic differentiation within the tissue and implications for clinical translation.

Cost-effective, expedited approaches for bone regeneration are urgently needed in an ageing population. Bone Morphogenetic Proteins (BMPs) stimulate osteogenesis but their efficacy is impeded by their short half-life. Delivery by genetically modified cells can overcome this problem. However, cell isolation and propagation represent significant obstacles for the translation into the clinic. Instead, complete gene activated fragments of adipose tissue hold great potential for bone repair. Here, using an in-vitro culture system, we investigated whether adenoviral transduction with human BMP-2 can promote osteogenic differentiation within adipose tissue fragments. Osteoinduction in adipose tissue fragments was evaluated by quantitative reverse transcriptase polymerase chain reaction, immunohistology and histomorphometry. BMP-2 transduced adipose tissue synthesized BMP-2 protein over 30 days peaking by day six, which significantly promoted osteogenic differentiation as indicated by increased calcium depositions, up-regulation of bone marker genes, and bone-related protein expression. Our results demonstrate that cells within adipose tissue fragments can differentiate osteogenically after BMP-2 transduction of cells on the surface of the adipose tissue. BMP-2 gene activated adipose tissue represents an advanced osteo-regenerative biomaterial that can actively contribute to osteogenesis and potentially enable the development of a novel, cost-effective, one-step surgical approach to bone repair without the need for cell isolation.

The Induction of Bone Formation: The Translation Enigma.

A paradigmatic shift in the way of thinking is what bone tissue engineering science requires to decrypt the translation conundrum from animal models into human. The deductive work of Urist (1965), who discerned the principle of bone induction from the pioneering works of Senn, Huggins, Lacroix, Levander, and other bone regenerative scientists, provided the basis that has assisted future bone tissue regenerative scientists to extend the bone tissue engineering field and its potential uses for bone regenerative medicine in humans. However, major challenges remain that are preventing the formation of bone by induction clinically. Growing experimental evidence is indicating that bone inductive studies are non-translatable from animal models into a clinical environment. This is preventing bone tissue engineering from reaching the next phase in development. Countless studies are trying to discern how the formation of bone by induction functions mechanistically, so as to try and solve this enigmatic problem. However, are the correct questions being asked? Why do bone inductive animal studies not translate into humans? Why do bone induction principles not yield the same extent of bone formation as an autogenous bone graft? What are bone tissue engineering scientists missing? By critically re-assessing the past and present discoveries of the bone induction field, this review article attempts to re-discover the field of bone formation by induction, identifying some key features that may have been missed. These include a detailed library of all proteins in bones and their arrangement in the 3D superstructure of the bone together with some other important criteria not considered by tissue engineering scientists. The review therefore not only re-iterates possible avenues of research that need to be re-explored but also seeks to guide present and future scientists in how they assess their own research in light of experimental design and results. By addressing these issues bone formation by induction without autografts might finally become clinically viable.

Effectiveness of 3-dimensional shoulder ultrasound in the diagnosis of rotator cuff tears: A meta-analysis.

BACKGROUND: Numerous quantitatively based studies measuring the accuracy of 3D shoulder ultrasound (US) for the diagnosis of rotator cuff tears remain inconclusive. In order to determine how effective 3D shoulder US is for detecting rotator cuff tears, a meta-analysis was performed systematically. METHODS: Six electronic databases, PubMed/Medline, Embase, Cochrane Library, CNKI, VIP data, and Wanfang data, were utilized to retrieve articles praising the diagnostic value of 3D shoulder US for use in detecting rotator cuff tears. After screening and diluting out the articles that met inclusion criteria to be used for statistical analysis, the pooled evaluation indexes including sensitivity, specificity, and diagnostic odds ratio (DOR) as well as the summary receiver operating characteristic curve (SROC) were calculated utilizing Meta-Disc v.1.4. RESULTS: Screening determined that out of 4220, 7 studies involving a total of 282 patients were deemed viable for inclusion in the meta-analysis. The results of the analysis showed that the sensitivity and specificity were at 94% and 83%, respectively, with a DOR of 60.06, Q* index of 0.9058 and the area under SROC of 0.9609. Additionally, a satisfactory accuracy of 3D shoulder US was observed in detecting full- and partial-thickness rotator cuff tears. CONCLUSION: This meta-analysis suggests that 3D shoulder US is very effective and highly accurate to detect full-thickness rotator cuff tears, but may lack accuracy in the diagnosis of partial tears.

Redundancy and Molecular Evolution: The Rapid Induction of Bone Formation by the Mammalian Transforming Growth Factor-ß3 Isoform.

The soluble osteogenic molecular signals of the transforming growth factor-ß (TGF-ß) supergene family are the molecular bases of the induction of bone formation and postnatal bone tissue morphogenesis with translation into clinical contexts. The mammalian TGF-ß3 isoform, a pleiotropic member of the family, controls a vast array of biological processes including the induction of bone formation. Recombinant hTGF-ß3 induces substantial bone formation when implanted with either collagenous bone matrices or coral-derived macroporous bioreactors in the rectus abdominis muscle of the non-human primate Papio ursinus. In marked contrast, the three mammalian TGF-ßs do not initiate the induction of bone formation in rodents and lagomorphs. The induction of bone by hTGF-ß3/preloaded bioreactors is orchestrated by inducing fibrin-fibronectin rings that structurally organize tissue patterning and morphogenesis within the macroporous spaces. Induced advancing extracellular matrix rings provide the structural anchorage for hyper chromatic cells, interpreted as differentiating osteoblasts re-programmed by hTGF-ß3 from invading myoblastic and/or pericytic differentiated cells. Runx2 and Osteocalcin expression are significantly up-regulated correlating to multiple invading cells differentiating into the osteoblastic phenotype. Bioreactors pre-loaded with recombinant human Noggin (hNoggin), a BMPs antagonist, show down-regulation of BMP-2 and other profiled osteogenic proteins' genes resulting in minimal bone formation. Coral-derived macroporous constructs preloaded with binary applications of hTGF-ß3 and hNoggin also show down-regulation of BMP-2 with the induction of limited bone formation. The induction of bone formation by hTGF-ß3 is via the BMPs pathway and it is thus blocked by hNoggin. Our systematic studies in P. ursinus with translational hTGF-ß3 in large cranio-mandibulo-facial defects in humans are now requesting the re-evaluation of "Bone: formation by autoinduction" in primate models including humans.

The synergistic induction of bone formation by the osteogenic proteins of the TGF-ß supergene family.

The momentum to compose this Leading Opinion on the synergistic induction of bone formation suddenly arose when a simple question was formulated during a discussion session on how to boost the often limited induction of bone formation seen in clinical contexts. Re-examination of morphological and molecular data available on the rapid induction of bone formation by the recombinant human transforming growth factor-ß3 (hTGF-ß3) shows that hTGF-ß3 replicates the synergistic induction of bone formation as invocated by binary applications of hOP-1:hTGF-ß1 at 20:1 by weight when implanted in heterotopic sites of the rectus abdominis muscle of the Chacma baboon, Papio ursinus. The rapid induction of bone formation in primates by hTGF-ß3 may stem from bursts of cladistic evolution, now redundant in lower animal species but still activated in primates by relatively high doses of hTGF-ß3. Contrary to rodents, lagomorphs and canines, the three mammalian TGF-ß isoforms induce rapid and substantial bone formation when implanted in heterotopic rectus abdominis muscle sites of P. ursinus, with unprecedented regeneration of full thickness mandibular defects with rapid mineralization and corticalization. Provocatively, thus providing potential molecular and biological rationales for the apparent redundancy of osteogenic molecular signals in primates, binary applications of recombinant human osteogenic protein-1 (hOP-1) with low doses of hTGF-ß1 and -ß3, synergize to induce massive ossicles in heterotopic rectus abdominis, orthotopic calvarial and mandibular sites of P. ursinus. The synergistic binary application of homologous but molecularly different soluble molecular signals has indicated that per force several secreted molecular signals are required singly, synchronously and synergistically to induce optimal osteogenesis. The morphological hallmark of the synergistic induction of bone formation is the rapid differentiation of large osteoid seams enveloping haematopoietic bone marrow that forms by day 15 in heterotopic rectus abdominis sites. Synergistic binary applications also induce the morphogenesis of rudimentary embryonic growth plates indicating that the "memory" of developmental events in embryo can be redeployed postnatally by the application of morphogen combinations. Synergistic binary applications or single relatively high doses of hTGF-ß3 have shown that hTGF-ß3 induces bone by expressing a variety of inductive morphogenetic proteins that result in the rapid induction of bone formation. Tissue induction thus invocated singly by hTGF-ß3 recapitulates the synergistic induction of bone formation by binary applications of hTGF-ß1 and -ß3 isoforms with hOP-1. Both synergistic strategies result in the rapid induction and expansion of the transformed mesenchymal tissue into large corticalized heterotopic ossicles with osteoblast-like cell differentiation at the periphery of the implanted reconstituted specimens with "tissue transfiguration" in vivo. Molecularly, the rapid induction of bone formation by binary applications of hOP-1 and hTGF-ß3 or by hTGF-ß3 applied singly resides in the up-regulation of selected genes involved in tissue induction and morphogenesis, Osteocalcin, RUNX-2, OP-1, TGF-ß1 and -ß3 with however the noted lack of TGF-ß2 up-regulation.

The induction of bone formation by the recombinant human transforming growth factor-ß3.

Implantation of recombinant human transforming growth factor-ß3 (hTGF-ß3) with coral-derived calcium carbonate-based macroporous bioreactors with limited conversion to hydroxyapatite (7% HA/CC) in the rectus abdominis muscle of the non-human primate Chacma baboon Papio ursinus induces endochondral bone formation. The exact mechanisms by which hTGF-ß3 signalling induces bone in heterotopic sites of P. ursinus are not known. Coral-derived 7% HA/CC bioreactors with and without 125 µg hTGF-ß3 were implanted in triplicate in the rectus abdominis muscle of 6 adult baboons. 7% HA/CC bioreactors either with or without hTGF-ß3 were loaded with 125 µg of recombinant human Noggin (hNoggin), a bone morphogenetic proteins (BMPs) antagonist. Tissues on day 15, 60 and 90 were analysed by histomorphometry and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Down-regulation of BMP-2 characterized 7% HA/CC constructs preloaded with 125 µg hNoggin with Noggin down-regulated on day 60 and 90 together with lack of TGF-ß3 expression. Down-regulation of BMP-2 correlated with minimal bone formation by induction. hTGF-ß3/hNoggin pre-treated bioreactors up-regulated BMP-2 but only on day 90 together with a significant down-regulation of Noggin on day 60 and 90, correlating with the induction of bone formation, albeit limited, on day 90 at the periphery of the macroporous bioreactors only. hTGF-ß3 treated bioreactors significantly down-regulated BMP-2 on day 15 whilst up-regulating BMP-2 on day 60 and 90, together with down-regulation of Noggin on day 60 and 90 correlating with the prominent induction of bone formation. hTGF-ß3 significantly up-regulated RUNX-2 and Osteocalcin expression on day 15 controlling the differentiation of progenitor stem cells into the osteoblastic lineage. The induction of bone as initiated by hTGF-ß3 in the rectus abdominis muscle of P. ursinus is via the BMPs pathway with hTGF-ß3 controlling the induction of bone formation by regulating the expression of BMPs via Noggin expression. These results unequivocally demonstrate that hTGF-ß3 elicits bone induction by up-regulation of endogenous BMP-2 and is blocked by hNoggin.

Synergistic induction of bone formation by hOP-1, hTGF-beta3 and inhibition by zoledronate in macroporous coral-derived hydroxyapatites.

Thirty coral-derived calcium carbonate-based macroporous constructs with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) were implanted in the rectus abdominis of three adult non-human primate Papio ursinus to investigate the intrinsic induction of bone formation. Macroporous constructs with 125 microg human recombinant osteogenic protein-1 (hOP-1) or 125 microg human recombinant transforming growth factor-beta(3) (hTGF-beta(3)) were also implanted. The potential synergistic interaction between morphogens was tested by implanting binary applications of hOP-1 and hTGF-beta(3) 5:1 by weight, respectively. To evaluate the role of osteoclastic activity on the implanted macroporous surfaces, coral-derived constructs were pre-loaded with 0.24 mg of bisphosphonate zoledronate (Zometa). To correlate the morphology of tissue induction with osteogenic gene expression and activation, harvested specimens on day 90 were analyzed for changes in OP-1 and TGF-beta(3) mRNA synthesis by quantitative real-time polymerase chain reaction (qRT-PCR). The induction of bone formation in 7% HA/CC solo correlated with OP-1 expression. Massive bone induction formed by binary applications of the recombinant morphogens. Single applications of hOP-1 and hTGF-beta(3) also resulted in substantial bone formation, not comparable however to synergistic binary applications. Zoledronate-treated macroporous constructs showed limited bone formation and in two specimens bone formation was altogether absent; qRT-PCR showed a prominent reduction of OP-1 gene expression whilst TGF-beta(3) expression was far greater than OP-1. The lack of bone formation by zoledronate-treated specimens indicates that osteoclastic activity on the implanted coral-derived constructs is critical for the spontaneous induction of bone formation. Indirectly, zoledronate-treated samples showing lack of OP-1 gene expression and absent or very limited bone formation by induction confirm that the spontaneous induction of bone formation by coral-derived macroporous constructs is initiated by secreted BMPs/OPs, in context the OP-1 isoform.