Bone Induction Research Articles

Hydroxyapatite coating affects the Wnt signaling pathway during peri-implant healing in vivo

Owing to its bio- and osteoconductivity, hydroxyapatite (HA) is a widely used implant material, but its osteogenic properties are only partly evaluated in vitro and in vivo. The present study focused on bone healing adjacent to HA-coated titanium (Ti) implants, with or without incorporated lithium ions (Li+). Special attention was given to the Wnt signaling pathway. The implants were inserted into rat tibia for 7 or 28days and analyzed ex vivo, mainly by histomorphometry and quantitative real-time polymerase chain reaction (qPCR). HA-coated implants showed, irrespective of Li+ content, bone–implant contact (BIC) and removal torque values significantly higher than those of reference Ti. Further, the expression of OCN, CTSK, COL1A1, LRP5/6 and WISP1 was significantly higher in implant-adherent cells of HA-coated implants, with or without Li+. Significantly higher β-catenin expression and significantly lower COL2A1 expression were observed in peri-implant bone cells from HA with 14ngcm−2 released Li+. Interestingly, Ti implants showed a significantly larger bone area (BA) in the threads than HA with 39ngcm−2 released Li+, but had a lower BIC than any HA-coated implant. This study shows that HA, with or without Li+, is a strong activator of the Wnt signaling pathway, and may to some degree explain its high bone induction capacity.

Evolution of a developmental mechanism: Species-specific regulation of the cell cycle and the timing of events during craniofacial osteogenesis

Neural crest mesenchyme (NCM) controls species-specific pattern in the craniofacial skeleton but how this cell population accomplishes such a complex task remains unclear. To elucidate mechanisms through which NCM directs skeletal development and evolution, we made chimeras from quail and duck embryos, which differ markedly in their craniofacial morphology and maturation rates. We show that quail NCM, when transplanted into duck, maintains its faster timetable for development and autonomously executes molecular and cellular programs for the induction, differentiation, and mineralization of bone, including premature expression of osteogenic genes such as Runx2 and Col1a1. In contrast, the duck host systemic environment appears to be relatively permissive and supports osteogenesis independently by providing circulating minerals and a vascular network. Further experiments reveal that NCM establishes the timing of osteogenesis by regulating cell cycle progression in a stage- and species-specific manner. Altering the time-course of D-type cyclin expression mimics chimeras by accelerating expression of Runx2 and Col1a1. We also discover higher endogenous expression of Runx2 in quail coincident with their smaller craniofacial skeletons, and by prematurely over-expressing Runx2 in chick embryos we reduce the overall size of the craniofacial skeleton. Thus, our work indicates that NCM establishes species-specific size in the craniofacial skeleton by controlling cell cycle, Runx2 expression, and the timing of key events during osteogenesis.

Zinc in calcium phosphate mediates bone induction: In vitro and in vivo model

Zinc-containing tricalcium phosphate (Zn-TCP) was synthesized to investigate the role of zinc in osteoblastogenesis, osteoclastogenesis and in vivo bone induction in an ectopic implantation model. Zinc ions were readily released in the culture medium. Zn-TCP with the highest zinc content enhanced the alkaline phosphatase activity of human bone marrow stromal cells and tartrate-resistant acid phosphatase activity, as well as multinuclear giant cell formation of RAW264.7 monocyte/macrophages. RAW264.7 cultured with different dosages of zinc supplements in medium with or without zinc-free TCP showed that zinc could influence both the activity and the formation of multinuclear giant cells. After a 12-week implantation in the paraspinal muscle of canines, de novo bone formation and bone incidence increased with increasing zinc content in Zn-TCP – up to 52% bone in the free space. However, TCP without zinc induced no bone formation. Although the observed bone induction cannot be attributed to zinc release alone, these results indicate that zinc incorporated in TCP can modulate bone metabolism and render TCP osteoinductive, indicating to a novel way to enhance the functionality of this synthetic bone graft material.

Ovatodiolide inhibits the maturation of allergen-induced bone marrow-derived dendritic cells and induction of Th2 cell differentiation

Ovatodiolide was a unique macrocyclic diterpenoid isolated from the traditional Chinese medicinal herb Anisomeles indica. The present study attempted to examine the ovatodiolide effects on dendritic cell (DC) maturation and immuno-stimulatory activities. The effects of ovatodiolide on DC surface molecule expression, cytokine production, and capacity to induce T-cell differentiation were examined in ovalbumin (OVA)/thymic stromal lymphopoietin (TSLP)-stimulated DCs. Ovatodiolide attenuated the expression of DC surface molecules CD80, CD86, histocompatibility complex (MHC) class II, and Th2 subset of CD4(+) T cells co-stimulatory molecule-OX40 ligand production. Additionally, ovatodiolide suppressed the CD4(+) T cells proliferation, and production of inflammatory cytokines interleukin (IL)-4, IL-5, and tumor necrosis factor (TNF)-α. This study may be useful to develop ovatodiolide as a therapeutic adjuvant.

Somitic disruption of GNAS in chick embryos mimics progressive osseous heteroplasia

Progressive osseous heteroplasia (POH) is a rare developmental disorder of heterotopic ossification (HO) caused by heterozygous inactivating germline mutations in the paternal allele of the GNAS gene. Interestingly, POH lesions have a bewildering mosaic distribution. Using clinical, radiographic, and photographic documentation, we found that most of the 12 individuals studied had a lesional bias toward one side or the other, even showing exclusive sidedness. Most strikingly, all had a dermomyotomal distribution of HO lesions. We hypothesized that somatic mutations in a progenitor cell of somitic origin may act on a background of germline haploinsufficiency to cause loss of heterozygosity at the GNAS locus and lead to the unilateral distribution of POH lesions. Taking advantage of the chick system, we examined our hypothesis by mimicking loss of heterozygosity of GNAS expression using dominant-negative GNAS that was introduced into a subset of chick somites, the progenitors that give rise to dermis and muscle. We observed rapid ectopic cartilage and bone induction at the axial and lateral positions in a unilateral distribution corresponding to the injected somites, which suggests that blocking GNAS activity in a targeted population of progenitor cells can lead to mosaic ectopic ossification reminiscent of that seen in POH.

Repair of a root perforation by using MTA: a case report

Root perforations are accidental events that may occur during the treatment, causing tissue inflammation and alveolar bone loss of integrity of the periodontium. In such cases, the radiological evidence is fundamental in the formulation of the diagnosis, in the choice of therapy (surgical or non-surgical) and finally for the assessment of prognosis of the dental element. In non-surgical treatment of endodontic lesions, the material used for the repair of the defect root should have biocompatibility, antibacterial activity, ability to induce healing of periodontal tissues and radiopacity. The Mineral Trioxide Aggregate (MTA) is a silicate-based cement introduced in dental clinical practice with good radiopacity, biocompatibility and bone induction. This article describes the use of MTA in endodontic repair of a perforation of the middle third root and the success of non-surgical treatment was dimonstrated radiographicaly.

Surface Modification and Coating of the Carbon/Carbon Composite in the Medical Fields

Carbon/Carbon composite is considered to be one of the best biomaterials substitute to human hard tissues due to its excellent biocompatibility and the much closed elastic modulus to human skeleton. It has been widely used and studied to the fields of artificial bones materials, but the osteoinductivity need to be improved. In recent years, the commonly used surface modification to improve their bone induction, such as hydroxyapatite, chitosan and so on, which prompted the adsorption of osseous protein, adhesion and growth of cells. It is believed the surface modification and coating of the carbon/carbon composite should promote its application in artificial bones. This article reviews the modified coat of medical carbon / carbon composites in recent years, and proposed some recommendations for the development of medical carbon / carbon composite in the future.

Abstract 672: Histone deacetylase inhibition has potential to suppresse growth of androgen dependent and independent prostate cancer in vitro and in vivo.

Abstract Histone acetylation plays an important role in chromatin remodeling and gene expression. Histone deacetylase (HDAC) inhibitors regulate gene expressions through the induction of histone acetylation and have been reported to suppress growth of several malignancies including lymphoma. In this study, we examined the effects of one example on androgen dependent and independent prostate cancer growth in vivo and in vitro. In in vitro study, androgen dependent and independent prostate cancer cell lines (LNCaP and VCaP) were used. The HDAC inhibitor, OBP-801 (Generously donated by Dr. Urata, Oncolys Biopharma), significantly suppressed cell proliferation with induction of G2/M arrest, and decreased expression of cdc2, androgen receptor (AR) and PSA protein in both cell lines. OBP-801 also significantly induced apoptosis with activation of caspase 3 in LNCaP. Next, we performed two in vivo experiments. First, we transplanted an androgen independent rat prostate cancer (PLS-P) into the femurs and backs of male F344 rats, and OBP-801 was administered intravenously at doses of 0, 2.5 and 5mg/kg every other day for 2 weeks. OBP-801 significantly reduced the volume of the transplanted tumor, while inducing cleaved caspase 3 protein expression. OBP-801 also suppressed bone destruction and induction of osteoclasts in xenografts transplanted to femurs. Second, we prepared male Transgenic Rat for Adenocarcinoma of Prostate (TRAP) rats which spontaneously develop androgen dependent rat prostate cancers, and OBP-801 was given intravenously at doses of 0, 0.5 and 2mg/kg once a week for 8 weeks. As a result, body weight loss was observed in treated groups, and numbers of carcinoma acini in the lateral and ventral prostates were significantly decreased compared with the control group values. In conclusion, the obtained data suggests that OBP-801 may be a candidate therapeutic or preventing agent for androgen dependent and independent prostate cancer, although side effects such as body weight loss may occur. Elucidation of mechanisms of HDAC underlying these effects is needed. Citation Format: Shinya Sato, Shugo Suzuki, Aya Naiki-Ito, Yoriko Yamashita, Makoto Asamoto, Tomoyuki Shirai, Satoru Takahashi. Histone deacetylase inhibition has potential to suppresse growth of androgen dependent and independent prostate cancer in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 672. doi:10.1158/1538-7445.AM2013-672

Bone induction by biomimetic PLGA copolymer loaded with a novel synthetic RADA16-P24 peptide in vivo

Bone morphogenetic protein-2 (BMP-2) is a key bone morphogenetic protein, and poly(lactic-co-glycolic acid) (PLGA) has been widely used as scaffold for clinical use to carry treatment protein. In the previous studies, we have synthesized BMP-2-related peptide (P24) and found its capacity of inducing bone regeneration. In this research, we have synthesized a new amphiphilic peptide Ac-RADA RADA RADA RADA S[PO4]KIPKASSVPTELSAISTLYLDDD-CONH2 (RADA16-P24) with an assembly peptide RADA16-Ion the P24 item of BMP2 to form divalent ion-induced gelatin. Two methods of physisorption and chemical cross-linking were used to bind RADA16-P24 onto the surface of the copolymer PLGA to synthesize RADA16-P24–PLGA, and its capacity of attaching bone marrow stromal cells (BMSCs) was evaluated in vitro and inducing ectopic bone formation was examined in vivo. In vitro our results demonstrated that RADA16-P24–PLGA copolymer prepared by physisorbing or prepared by chemical cross-linking had a peptide binding rate of (2.0180±0.5296)% or (10.0820±0.8405)% respectively (P<0.05). In addition the BMSCs proliferated vigorously in the RADA16-P24–PLGA biomaterials. Significantly the percentage of BMSCs attached to RADA16-P24–PLGA composite prepared by chemical cross-linking and physisorbing were (71.4±7.5) % or (46.7±5.8) % (P<0.05). The in vivo study showed that RADA16-P24–PLGA chemical cross-linking could better induce ectopic bone formation compared with RADA16-P24–PLGA physisorbing and PLGA. It is concluded that the PLGA copolymer is a good RADA16-P24 carrier. This novel RADA16-P24–PLGA composite has strong osteogenic capability.

Bone induction by surface-double-modified true bone ceramics in vitro and in vivo

True bone ceramic (TBC), obtained by twice sintering fresh bovine cancellous bone at high temperatures, is an osteoconductive and bioactive bone substitute material that exhibits excellent biocompatibility with hard tissue. The authors have previously synthesized a novel BMP-2-related peptide, P24, and found that it could enhance the osteoblastic differentiation of cells. The objective of the present study was to construct a double-modified TBC via mineralization into simulated body fluid and P24 incorporation for enhanced bone formation. In vitro experiments revealed that surface mineralization-modified (SMM) TBC scaffolds demonstrated efficiency for sustained release of P24. The P24/SMM-TBC composite exhibited increased osteogenic activity by cell adhesion rate determination, MTT assay, alkaline phosphatase staining, and calcium nodule staining with alizarin red compared with SMM-TBC and TBC. In vivo studies showed that the P24/SMM-TBC composite scaffold promoted significant bone defect repair, in marked contrast to stand-alone SMM-TBC and TBC, based on the results of radiographic evaluation and histological examination. These findings indicate that SMM-TBC is a good scaffold for the controlled release of P24 and that the P24/SMM-TBC composite could improve the adhesion, proliferation and differentiation of cells and repair bone defects. The double-modified P24/SMM-TBC composite biomaterial shows potential for clinical application in bone tissue engineering.

Induction of craniofacial bone from mouse embryonic stem cells

Induction of craniofacial bone from mouse embryonic stem cells

Osteogenic Efficiency of Demineralized and Lyophylized Xenogeneic Bone and Syngeneic Dentine Implants in Mice

Intramuscular implantation of demineralized and lyophilized rat bone matrix and murine lower incisors into thigh muscles of BALB/c mice results in deposits of bone adjacent to the implants, a phenomenon termed as ectopic osteogenesis. The yield of induced bone does not critically depend on the mass of implanted matrices, and thus on the quantity of bone morphogenetic proteins (BMPs) present in the implants. A positive correlation between bone matrix implant weight and the yield of induced bone was observed only 28 days post grafting, i.e. when endochondreal osteogenesis is completed and bone resorption has not advanced. A more consistent yield of bone induction wasobserved in the case of demineralized tooth implants. It is postulated that chondro/osteoinduction by demineralized, lyophilized matrix implants is not determined by the range of BMPs presumably released in proportion to implant size, but is rather limited by the population of responsive host mesenchymal cells.

Zirconium, calcium, and strontium contents in magnesium based biodegradable alloys modulate the efficiency of implant-induced osseointegration

Development of new biodegradable implants and devices is necessary to meet the increasing needs of regenerative orthopedic procedures. An important consideration while formulating new implant materials is that they should physicochemically and biologically mimic bone-like properties. In earlier studies, we have developed and characterized magnesium based biodegradable alloys, in particular magnesium-zirconium (Mg-Zr) alloys. Here we have reported the biological properties of four Mg-Zr alloys containing different quantities of strontium or calcium. The alloys were implanted in small cavities made in femur bones of New Zealand White rabbits, and the quantitative and qualitative assessments of newly induced bone tissue were carried out. A total of 30 experimental animals, three for each implant type, were studied, and bone induction was assessed by histological, immunohistochemical and radiological methods; cavities in the femurs with no implants and observed for the same period of time were kept as controls. Our results showed that Mg-Zr alloys containing appropriate quantities of strontium were more efficient in inducing good quality mineralized bone than other alloys. Our results have been discussed in the context of physicochemical and biological properties of the alloys, and they could be very useful in determining the nature of future generations of biodegradable orthopedic implants.

Bone morphogenetic proteins: Periodontal regeneration

Periodontitis is an infectious inflammatory disease that results in attachment loss and bone loss. Regeneration of the periodontal tissues entails de novo formation of cementum, periodontal ligament, and alveolar bone. Several different approaches are currently being explored to achieve complete, reliable, and reproducible regeneration of periodontal tissues. The therapeutic management of new bone formation is one of the key issues in successful periodontal regeneration. Bone morphogenetic proteins form a unique group of proteins within the transforming growth factor superfamily of genes and have a vital role in the regulation in the bone induction and maintenance. The activity of bone morphogenetic proteins was first identified in the 1960s, but the proteins responsible for bone induction were unknown until the purification and cloning of human bone morphogenetic proteins in the 1980s, because of their osteoinductive potential. Bone morphogenetic proteins have gained a lot of interest as therapeutic agents for treating periodontal defects. A systematic search for data related to the use of bone morphogenetic proteins for the regeneration of periodontal defects was performed to recognize studies on animals and human (PUBMED, MEDLINE, COCHRANE, and Google search). All the studies included showed noticeable regeneration of periodontal tissues with the use of BMP.

Cyclosporin A Enhances Callus Formation in Rabbit Tibia Fractures

Purpose: Drugs that modify the production of cytokines may affect fracture healing.The immunosuppressive drug cyclosporin A is widely used to modify the immune response in transplantations and in treatment of rheumatoid disorders. We wanted to analyze the effect of cyclosporin A on fracture healing and on the development of trauma induced osteopenia.Methods: Experimental tibia fractures were stabilised with intramedullary pins in 26 rabbits. The animals were given 5mg/kg/day of cyclosporin A or placebo for 5 weeks. Bone mineral content, callus volume and biomechanical testing were performed on both tibias and femurs.Results: At 5 weeks cyclosporin A treatment resulted in increased bone mineral content and increased callus volume of the fractured bone. The femora on the operated side had significantly lower bone mineral content compared to the non-operated side. This trauma induced osteopenia was unaffected by cyclosporin A treatment. Failure torque and stiffness of the tibia and femora were similar in both groups.Interpretation: Cyclosporin A stimulates bone formation in fracture repair. The mechanism is unclear, but a direct or cytokine mediated effect on bone forming cells, or enhanced bone induction resulting in increased bone formation, is possible.

The effects of angiotensin II and the oxidative stress mediator 4-hydroxynonenal on human osteoblast-like cell growth: possible relevance to otosclerosis

Otosclerosis is a complex disease characterized by an abnormal bone turnover of the otic capsule resulting in conductive hearing loss. Recent findings have shown that angiotensin II (Ang II), a major effector peptide of the renin–angiotensin system, plays an important role in the pathophysiology of otosclerosis, most likely by its proinflammatory effects on the bone cells. Because reactive oxygen species play a role both in inflammation and in the cellular signaling pathway of Ang II, the appearance of protein adducts of the “second messenger of free radicals,” the aldehyde 4-hydroxynonenal (HNE), in otosclerotic bone has been analyzed. Immunohistochemical analysis of HNE-modified proteins in tissue samples of the stapedial bones performed on 15 otosclerotic patients and 6 controls revealed regular HNE–protein adducts present in the subperiosteal parts of control bone specimens, whereas irregular areas of a pronounced HNE–protein adduct presence were found within stapedial bone in cases of otosclerosis. To study possible interference by HNE and Ang II in human bone cell proliferation, differentiation, and induction of apoptosis we used an in vitro model of osteoblast-like cells. HNE interacted with Ang II in a dose-dependent manner, both by forming HNE–Ang II adducts, as revealed by immunoblotting, and by modifying its effects on cultured cells. Namely, treatment with 0.1nM Ang II and 2.5μM HNE stimulated proliferation, whereas treatment with 10μM HNE or in combination with Ang II (0.1, 0.5, and 1nM) decreased cell proliferation. Moreover, 10μM HNE alone and with Ang II (except if 1nM Ang II was used) increased cellular differentiation and apoptosis. HNE at 5μM did not affect differentiation nor significantly change apoptosis. On the other hand, when cells were treated with lower concentrations of HNE and Ang II we observed a decrease in cellular differentiation (combination of 1.0 or 2.5μM HNE with 0.1nM Ang II) and decrease in apoptosis (0.1 and 0.5nM Ang II). Cellular necrosis was increased with 5 and 10μM HNE if given alone or combined with Ang II, whereas 0.5nM Ang II and combination of 1μM HNE with Ang II (0.1 and 0.5nM) reduced necrosis. These results indicate that HNE and Ang II might act mutually dependently in the regulation of bone cell growth and in the pathophysiology of otosclerosis.

Atrophic femoral nonunion successfully treated with teriparatide

Teriparatide is a synthetic polypeptide hormone that contains the 1-34 aminoacid fragment of the recombinant human parathyroid hormone. It has been approved for the treatment of postmenopausal women with osteoporosis who are at high risk for sustaining a fragility fracture. It has been shown that teriparatide also accelerates fracture healing by improving the biomechanical properties of the fracture callus, increasing endochondral ossification and bone remodeling in animal models. This effect has been observed in several case reports. Fracture healing disorders negatively affect the patient's quality of life and result in high healthcare costs, as a second surgery is required to stabilize the fracture and stimulate bone biology. Future biotechnologies that accelerate fracture healing may be useful tools. We present a case report of delayed union of a femoral fracture treated with teriparatide. She was diagnosed with right distal metaphyseal femoral fracture on total knee arthroplasty. She underwent surgery at our center consisting of ORIF with lateral femoral locking plate in October 2011. Radiologic controls at 5 and 7 months did not show any signs of healing. After 2 months of treatment with teriparatide, the X-ray showed the presence of bone bridges and a decreased gap between fragments and a different aspect of neoformed bone. After 3 months of treatment, healing was complete. Our case report seems to confirm the possible effect of TPTD as bone induction through a more rapid healing of fractures. The TPTD could have a potentially important role in treating some forms of nonunion and delay in consolidation. Thus, one could hypothesize the possibility of a medical treatment with TPTD both as a preventive way and also as a support to the synthesis in high risk of nonunion fractures and complexed fractures in osteoporotic bone.

Characteristics of Surface Behavior and Osteoinductivity of Biomimetic Ceramic Scaffold

We previously reported functionally graded hydroxyapatite (fg-HAp) with the characteristics of blood permeability into the bulk and osteoinduction by adding low dose of bone morphogenetic protein-2 (BMP-2). In this study, we evaluated the bioactivity of the obtained dense HAp bodies in a simulated body fluid (SBF) and the osteoinductive activity with or without SBF treatment in vivo. The fg-HApSBF was prepared by immersing the fg-HAp in the SBF solution at 14 days and the surface structure was observed by SEM. The fg-HApSBF showed fine bone-like crystal on the surface of the HAp. Ectopic bone formation occurred in the fg-HAp/BMP-2 (1.0, 0.5μg) system at 3 weeks, while only in the fg-HApSBF/BMP-2 (1.0μg), bone induction was found. The histological finding showed body fluid permeation into the fg-HApSBF bulk and bone formation were obserbed.

Bone tissue engineering of induced pluripotent stem cells cultured with macrochanneled polymer scaffold

A reliable source of osteogenic cells is an essential factor for bone tissue engineering. In this study, human-induced pluripotent stem cells (hiPSCs) without an embryoid body step were cultured in macrochanneled poly(caprolactone) (PCL) scaffolds prepared using a robotic dispensing technique, after which osteogenesis was promoted by the addition of exogenous osteogenic factors. The osteogenesis of the hiPSCs was demonstrated based on the detection of osteogenic molecules, such as osteopontin, using flow cytometry analysis, quantitative polymerase chain reaction and western blotting. Thereafter, the cell-scaffold constructs were transplanted into the subcutaneous site of male athymic mice. At 4 weeks after implantation, histological assays (hematoxylin & eosin staining, Alizarin red staining, and osteocalcin immunostaining) were conducted to determine the bone induction of hiPSCs. The results indicated a production of pronounced levels of extracellular matrices and their mineral deposition within the cell-scaffold implant, suggesting possible in vivo bone induction by the hiPSCs-based tissue engineering approach. The results presented here provide useful information regarding the tissue engineering of bone utilizing hiPSCs in conjunction with cell-supporting scaffolds.

Critical assessment of rhBMP-2 mediated bone induction: An in vitro and in vivo evaluation

Understanding the influence of formulation and storage conditions on rhBMP-2 bioactivity is extremely important for its clinical application. Reports in the literature show that different research groups employ different parameters such as formulation conditions, storage, doses for in vivo applications etc. that makes it difficult to correlate results from different experiments. We therefore decided to rationalize these anomalies by performing a basic study on such parameters using two commercially available BMPs. Our in vitro experiments suggest that BMPs from different sources have significant differences in their bioactivity. The clinically approved rhBMP-2 (InductOs®; BMP-P) showed superior stability, compared to rhBMP-2 from R&D Systems (BMP-R) at physiological pH (determined by ALP assay). This BMP-P also showed lower binding to polypropylene Eppendorf tube. The BMP-R almost lost its bioactivity within 30min at physiological pH and also shows more adhesion to plastic surfaces. This aggregation behavior was unequivocally ascertained by performing light scattering studies of the two BMPs, which revealed linear aggregation with time for BMP-R unlike BMP-P. The in vitro results were also reflected in the in vivo experiments, in a rat ectopic model with injectable hyaluronic acid (HA) hydrogel as BMP carrier. After 7weeks post-implantation we observed larger bone volume with oriented collagen in the BMP-P group but a smaller bone with disoriented collagen in the BMP-R case. Our results highlight the large difference in activity between seemingly identical substances and also the importance of proper handling of such sensitive proteins.