Substance P increases CCN2 dependent on TGF-beta yet Collagen Type I via TGF-beta1 dependent and independent pathways in tenocytes

Transforming growth factor beta 1 (TGFbeta-1) and connective tissue growth factor (CCN2) are important mediators of tissue repair and fibrosis, with CCN2 functioning as a downstream mediator of TGFβ-1. Substance P (SP) is also linked to collagen production in tenocytes. A link between SP, TGFbeta-1 and CCN2 has yet to be established in tenocytes or fibrogenic processes. We sought to determine whether SP induces tenocyte proliferation, CCN2, or collagen production via TGFbeta-1 signaling or independently in rat primary tenocytes. Tenocytes were isolated from rat tendons, cultured and stimulated by SP and/or TGFbeta-1. Cultured cells expressed proteins characteristic of tenocytes (vimentin and tenomodulin) and underwent increased proliferation dose dependently after SP and TGFbeta-1 treatments, alone or combined (more than SP alone when combined). SP induced TGFbeta-1 expression in tenocytes in both dose- and time-dependent manners. SP and TGFbeta-1, alone or combined, stimulated CCN2 expression in tenocytes and their supernatants after both 24 and 48 h of stimulation; a response blocked with addition of a TGFbeta-1 receptor inhibitor. In contrast, SP potentiated collagen type I secretion by tenocytes, a response abrogated by the TGFbeta-1 receptor inhibitor after 48 h of stimulation, but not after the shorter 24 h of stimulation. Our findings suggest that both SP and TGFbeta-1 can stimulate tenocyte fibrogenic processes, albeit differently. TGFbeta-1 pathway signaling was involved in CCN2 production at all time points examined, while SP induced collagen type I production independently prior to the onset of signaling through the TGFbeta-1 pathway.     

In vivo tendon engineering with skeletal muscle derived cells in a mouse model

Engineering a functional tendon with strong mechanical property remains an aim to be achieved for its eventual application. Both skeletal muscle and tendon are closely associated during their development and both can bear strong mechanical loading dynamically. This study explored the possibility of engineering stronger tendons with mouse skeletal muscle derived cells (MDCs) and with mouse tenocytes as a control. The results demonstrated that both MDCs and tenocytes shared the gene expression of growth differentiation factor-8 (GDF-8), collagens I, III, VI, scleraxis and tenomodulin, but with MyoD gene expression only in MDCs. Quantitatively, MDCs expressed higher levels of GDF-8, collagens III and VI (p < 0.05), whereas tenocytes expressed higher levels of collagen I, scleraxis and tenomodulin (p < 0.05). Interestingly, MDCs proliferated faster with more cells in S + G2/M phases than tenocytes (p < 0.05). After been seeded on polyglycolic acid (PGA) fibers, MDCs formed better quality engineered tendons with more mature collagen structure and thicker collagen fibrils as opposed to tenocyte engineered tendons. Biochemically, more collagen VI and decorin were produced in the former than in the later. Functionally, MDC engineered tendons exhibited stronger mechanical properties than tenocyte engineered tendons, including maximal load, stiffness, tensile strength and Young's modulus (p < 0.05). Furthermore, with the increase of implantation time, MDCs gradually lost their expression of myogenic molecules of MyoD and desmin and gained the expression of tenomodulin, a marker for tenocytes. Collectively, these results indicate that MDCs may serve as a desirable alternative cell source for engineering functional tendon tissue.     

The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities

Adipose-derived stem cells (ASCs) have valuable applications in regenerative medicine, but maintaining the stemness of ASCs during in vitro culture is still a challenging issue. In this study, human ASCs spontaneously formed three-dimensional spheroids on chitosan films. Most ASCs within the spheroid were viable, and the cells produced more extracellular molecules, like laminin and fibronectin. Comparing to monolayer culture, ASC spheroids also exhibited enhanced cell survival in serum deprivation condition. Although cell proliferation was inhibited in spheroids, ASCs readily migrated out and proliferated upon transferring spheroids to another adherent growth surface. Moreover, spheroid-derived ASCs exhibited higher expansion efficiency and colony-forming activity. Importantly, we demonstrated that spheroid formation of human ASCs on chitosan films induced significant upregulation of pluripotency marker genes (Sox-2, Oct-4 and Nanog). By culturing the ASC spheroids in proper induction media, we found that ASC differentiation capabilities were significantly enhanced after spheroid formation, including increased transdifferentiation efficiency into neuron and hepatocyte-like cells. In a nude mice model, we further showed a significantly higher cellular retention ratio of ASC spheroids after intramuscular injection of spheroids and dissociated ASCs. These results suggested that ASCs cultured as spheroids on chitosan films can increase their therapeutic potentials.     

Differences in morphogenesis of 3D cultured primary human osteoblasts under static and microfluidic growth conditions

As information on osteoblast mechanosensitivity response to biomechanical cues in three-dimensional (3D) in vitro microenvironments is sparse, the present study compared morphogenesis of primary human alveolar bone osteoblasts (PHABO) under microchip-based 3D-static conditions, and 3D-fluid flow-mediated biomechanical stimulation in perfusion bioreactors. Discrimination of the respective microenvironment by differential morphogenesis was evident from fluid flow-induced PHABO reorganization into rotund bony microtissue, comprising more densely packed multicellular 3D-aggregates, while viability of microtissues was flow rate dependent. Time-lapse microscopy and simple modeling of biomechanical conditions revealed that physiologically relevant fluid flow-mediated PHABO stimulation was associated with formation of mulberry-like PHABO aggregates within the first 24 h. Differential extracellular matrix deposition patterns and gene expression modulation in PHABO aggregates at day 7 further indicates progressive osteoblast differentiation exclusively in perfusion culture-developed bony microtissues. The results of our study strongly suggest PHABO morphogenesis as discriminator of microenvironmental growth conditions, which in case of the microfluidic 3D microchip-bioreactor are substantiated by triggering in vitro bone microtissue formation concomitant with progressive osteoblastic differentiation. Such microtissue outcomes provide unique insight for mechanobiological studies in response to biomechanical fluid flow cues, and clinically appear promising for in vitro PHABO preconditioning, enabling innovative bone augmentation procedures.     

The crucial role of mechanical heterogeneity in regulating follicle development and ovulation with engineered ovarian microtissue

Contemporary systems for in vitro culture of ovarian follicles do not recapitulate the mechanical heterogeneity in mammalian ovary. Here we report microfluidic generation of biomimetic ovarian microtissue for miniaturized three-dimensional (3D) culture of early secondary preantral follicles by using alginate (harder) and collagen (softer) to fabricate the ovarian cortical and medullary tissues, respectively. This biomimetic configuration greatly facilitates follicle development to antral stage. Moreover, it enables in vitro ovulation of cumulus–oocyte complex (COC) from the antral follicles in the absence of luteinizing hormone (LH) and epidermal growth factor (EGF) that are well accepted to be responsible for ovulation in contemporary literature. These data reveal the crucial role of mechanical heterogeneity in the mammalian ovary in regulating follicle development and ovulation. The biomimetic ovarian microtissue and the microfluidic technology developed in this study are valuable for improving in vitro culture of follicles to preserve fertility and for understanding the mechanism of follicle development and ovulation to facilitate the search of cures to infertility due to ovarian disorders.     

Substrate-mediated nanoparticle/gene delivery to MSC spheroids and their applications in peripheral nerve regeneration

Substrate-derived mesenchymal stem cell (MSC) spheroids show greater differentiation capacities than dispersed single cells in vitro. During spheroid formation, nanoparticles (NPs)/genes may be delivered into the cells. In this study, MSCs were conveniently labeled with superparamagnetic Fe₃O₄ NPs, or transfected with brain-derived neurotrophic factor (BDNF) gene, by the substrate-mediated NP/gene uptake. With the promising in vitro data showing the beneficial effect on neural development and neurotrophic factor expression, MSCs were combined with a polymeric nerve conduit to bridge a 10 mm transection gap of rat sciatic nerve. High-resolution (7-T) magnetic resonance imaging (MRI) was used to track the transplanted cells. Nerve regeneration was assessed by functional recovery and histology. Results revealed that Fe₃O₄ NP-labeled MSCs were successfully visualized by MRI in vivo. Animals receiving BDNF-transfected MSC spheroids demonstrated the shortest gap bridging time (<21 days), the largest regenerated nerve, and the thickest myelin sheath at 31 days. Compared to MSC single cells, the pristine or BDNF-transfected MSC spheroids significantly promoted the functional recovery of animals, especially for the BDNF-transfected MSC spheroids. The transplanted MSCs were incorporated in the regenerated nerve and differentiated into non-myelinating Schwann cells after 31 days. This study suggests that the substrate-mediated gene delivery and NP labeling may provide extra values for MSC spheroids to carry therapeutic/diagnostic agents in cell-based therapy.     

Nilotinib Treatment of Patients Affected by Chronic Graft-versus-Host Disease Reduces Collagen Production and Skin Fibrosis by Downmodulating the TGF-β and p-SMAD Pathway

The present study was conducted to investigate cellular and molecular features of chronic graft-versus-host disease fibroblasts (GVHD-Fbs) and to assess the effectiveness of nilotinib as a fibrosis modulator. Growth kinetics, phenotype, and differentiation of cultured skin biopsy-derived GVHD-Fbs were compared with normal fibroblasts from both a dermal cell line (n-Fbs) and healthy individuals undergoing cosmetic surgery (n-skin-Fbs). Collagen genes (COL1α1/COL1α2) and p-SMAD2 expression were assessed by real-time PCR and immunofluorescence. The in vivo effects of nilotinib on chronic GVHD (cGVHD)-affected skin were investigated by immunohistochemistry; the relationship to TGF-β plasma levels was assessed. Although the morphology, phenotype, and differentiation of cultured GVHD-Fbs were comparable to normal fibroblasts, growth was slower and senescence was reached earlier. The expression of COL1α1 and COL1α2 mRNAs was respectively 4 and 1.6 times higher in cGVHD-Fbs (P = .02); the addition of TGF-β increased n-Fbs, but not GVHD-Fbs, collagen gene expression. Compared with the baseline, the addition of 1 μM nilotinib induced 86.5% and 49% reduction in COL1α1 and COL1α2 expression in cultured GVHD-Fbs, respectively (P< .01). In vivo immunohistochemistry analysis of skin biopsy specimens from patients with cGVHD showed strong baseline staining for COL1α1 and COL1α2, which decreased sharply after 180 days of nilotinib; immunofluorescence revealed TGF-β inhibition and p-Smad2 reduction at the intracellular level. Of note, nilotinib treatment was associated with normalization of TGF-β levels both in culture supernatants and in plasma. In general, the data show that cGVHD fibroblasts promote fibrosis through abnormal collagen production induced by hyperactive TGF-β signaling. TGF-β inhibition at the intracellular and systemic level represents an essential antifibrotic mechanism of nilotinib in a clinical setting.     

Induction of transient tenogenic phenotype of high-density cultured human dermal fibroblasts

Previous study showed that high-density culture supported phenotype maintenance of in vitro expanded tenocytes. This study explored the possibility of inducing the tenogenic phenotype of dermal fibroblasts by high-density monolayer culture. Human fibroblasts were seeded either in high-density (2.5?×?10⁶ per 10?cm dish) or at low-density (0.36?×?10⁶ per 10?cm dish). A preliminary tenogenic phenotype was observed in high-density cultured cells after one passage with significantly enhanced tenogenic gene expression. With continued cultivation to passage 3, scleraxis (SCX), tenomodulin (TNMD), collagen I, III, VI, decorin and tenascin-c were all significantly upregulated in high-density cultured dermal fibroblasts as opposed to low-density cells. High-density culture also led to relatively elongated cell shape, whereas cells appeared in spread shape in low-density culture. In addition, cytochalasin D treatment disrupted the cellular cytoskeleton and resulted in inhibition of density-induced tenogenic gene expression. However, high-density cultured fibroblasts failed to induce other lineage differentiations (osteogenic, chondrogenic and adipogenic). It also failed to induce tenogenic phenotype in high-density cultured chondrocytes. Mechanism studies revealed enhanced gene expression of growth and differentiation factors (GDF) 5, 6, 7 and 8 and transforming growth factor-β (TGF-β)1 in the high-density group and enhanced protein production of both GDF8 and TGF-β1. Moreover, BMP/GDF signaling inhibitor (LDN193189) and TGF-β signaling inhibitor (LY2109761) could both abrogate the density induced phenotype. In conclusion, high-density culture was able to induce transient tenogenic phenotype of dermal fibroblasts likely via cell morphology change and production of pro-tenogenic factors.     

Modeling of cancer metastasis and drug resistance via biomimetic nano-cilia and microfluidics

Three-dimensional (3D) tissue culture platforms that are capable of mimicking in vivo microenvironments to replicate physiological conditions are vital tools in a wide range of cellular and clinical studies. Here, learning from the nature of cilia in lungs – clearing mucus and pathogens from the airway – we develop a 3D culture approach via flexible and kinetic copolymer-based chains (nano-cilia) for diminishing cell-to-substrate adhesion. Multicellular spheroids or colonies were tested for 3–7 days in a microenvironment consisting of generated cells with properties of putative cancer stem cells (CSCs). The dynamic and reversible regulation of epithelial–mesenchymal transition (EMT) was examined in spheroids passaged and cultured in copolymer-coated dishes. The expression of CSC markers, including CD44, CD133, and ABCG2, and hypoxia signature, HIF-1α, was significantly upregulated compared to that without the nano-cilia. In addition, these spheroids exhibited chemotherapeutic resistance in vitro and acquired enhanced metastatic propensity, as verified from microfluidic chemotaxis assay designed to replicate in vivo-like metastasis. The biomimetic nano-cilia approach and microfluidic device may offer new opportunities to establish a rapid and cost-effective platform for the study of anti-cancer therapeutics and CSCs.     

Clinical and genetic aspects of Ehlers-Danlos syndrome,classic type

Classic Ehlers-Danlos syndrome is a heritable connective tissue disorder characterized by skin hyperextensibility, fragile and soft skin, delayed wound healing with formation of atrophic scars, easy bruising, and generalized joint hypermobility. It comprises Ehlers-Danlos syndrome type I and Ehlers-Danlos syndrome type II, but it is now apparent that these form a continuum of clinical findings and differ only in phenotypic severity. It is currently estimated that approximately 50% of patients with a clinical diagnosis of classic Ehlers-Danlos syndrome harbor mutations in the COL5A1 and the COL5A2 gene, encoding the α1 and the α2-chain of type V collagen, respectively. However, because no prospective molecular studies of COL5A1 and COL5A2 have been performed in a clinically well-defined patient group, this number may underestimate the real proportion of patients with classic Ehlers-Danlos syndrome harboring a mutation in one of these genes. In the majority of patients with molecularly characterized classic Ehlers-Danlos syndrome, the disease is caused by a mutation leading to a nonfunctional COL5A1 allele and resulting in haploinsufficiency of type V collagen. A smaller proportion of patients harbor a structural mutation in COL5A1 or COL5A2, causing the production of a functionally defective type V collagen protein. Most mutations identified so far result in a reduced amount of type V collagen in the connective tissues available for collagen fibrillogenesis. Inter- and intrafamilial phenotypic variability is observed, but no genotype-phenotype correlations have been observed. No treatment for the underlying defect is presently available for Ehlers-Danlos syndrome. However, a series of preventive guidelines are applicable.     

Engineering human neo-tendon tissue in vitro with human dermal fibroblasts under static mechanical strain

Proper cell source is one of the key issues for tendon engineering. Our previous study showed that dermal fibroblasts could be used to successfully engineer tendon in vivo and tenocytes could engineer neo-tendon in vitro with static strain. This study further investigated the possibility of engineering human neo-tendon tissue in vitro using dermal fibroblasts. Human dermal fibroblasts were seeded on polyglycolic acid (PGA) fibers pre-fixed on a U-shape as a mechanical loading group, or simply cultured in a dish as a tension-free group. In addition, human tenocytes were also seeded on PGA fibers with tension as a comparison to human dermal fibroblasts. The results showed that human neo-tendon tissue could be generated using dermal fibroblasts during in vitro culture under static strain and the tissue structure became more mature with the increase of culture time. Longitudinally aligned collagen fibers and spindle shape cells were observed histologically and collagen fibril diameter and tensile strength increased with time and reached a peak at 14 weeks. In contrast, the dermal fibroblast-PGA constructs failed to form neo-tendon, but formed disorganized fibrous tissue in tension-free condition with significantly weaker strength and poor collagen fiber formation. Interestingly, neo-tendon tissues generated with human dermal fibroblasts were indistinguishable from the counterpart engineered with human tenocytes, which supports the viewpoint that human dermal fibroblasts is likely to replace tenocytes for future tendon graft development in vitro with dynamic mechanical loading in a bioreactor system.     

SOX trio-co-transduced adipose stem cells in fibrin gel to enhance cartilage repair and delay the progression of osteoarthritis in the rat

The aim of this study was to test the hypotheses that retroviral gene transfer of SOX trio enhances the in vitro chondrogenic differentiation of ASCs, and that SOX trio-co-transduced ASCs in fibrin gel promote the healing of osteochondral defects, and arrest the progression of surgically-induced osteoarthritis in a rat model. ASCs isolated from inguinal fat in rats were transduced with SOX trio genes using retrovirus, and further cultured in vitro in pellets for 21 days, then analyzed for gene and protein expression of SOX trio and chondrogenic markers. SOX trio-co-transduced ASCs in fibrin gel were implanted on the osteochondral defect created in the patellar groove of the distal femur, and also injected into the knee joints of rats with surgically-induced osteoarthritis. Rats were sacrificed after 8 weeks, and analyzed grossly and microscopically. After 21 days, ASCs transduced with SOX-5, -6, or -9 had hundreds-fold greater gene expression of each gene compared with the control with the SOX protein expression matching gene expression. SOX trio-co-transduction significantly increased GAG contents as well as type II collagen gene and protein expression. ASCs co-transduced with SOX trio significantly promoted the in vivo cartilage healing in osteochondral defect model, and prevented the progression of degenerative changes in surgically-induced osteoarthritis.     

The effects of spheroid formation of adipose-derived stem cells in a microgravity bioreactor on stemness properties and therapeutic potential

Adipose-derived stem cells (ADSCs) represent a valuable source of stem cells for regenerative medicine, but the loss of their stemness during in vitro expansion remains a major roadblock. We employed a microgravity bioreactor (MB) to develop a method for biomaterial-free-mediated spheroid formation to maintain the stemness properties of ADSCs. ADSCs spontaneously formed three-dimensional spheroids in the MB. Compared with monolayer culture, the expression levels of E-cadherin and pluripotent markers were significantly upregulated in ADSC spheroids. Spheroid-derived ADSCs exhibited increased proliferative ability and colony-forming efficiency. By culturing the spheroid-derived ADSCs in an appropriate induction medium, we found that the multipotency differentiation capacities of ADSCs were significantly improved by spheroid culture in the MB. Furthermore, when ADSCs were administered to mice with carbon tetrachloride-induced acute liver failure, spheroid-derived ADSCs showed more effective potentials to rescue liver failure than ADSCs derived from constant monolayer culture. Our results suggest that spheroid formation of ADSCs in an MB enhances their stemness properties and increases their therapeutic potential. Therefore, spheroid culture in an MB can be an efficient method to maintain stemness properties, without the involvement of any biomaterials for clinical applications of in vitro cultured ADSCs.     

Novel defects in collagen XII and VI expand the mixed myopathy/Ehlers–Danlos syndrome spectrum and lead to variant-specific alterations in the extracellular matrix

PurposeTo date, heterozygous or homozygous COL12A1 variants have been reported in 13 patients presenting with a clinical phenotype overlapping with collagen VI–related myopathies and Ehlers–Danlos syndrome (EDS). The small number of reported patients limits thorough investigation of this newly identified syndrome, currently coined as myopathic EDS.MethodsDNA from 78 genetically unresolved patients fulfilling the clinical criteria for myopathic EDS was sequenced using a next-generation panel of COL12A1, COL6A1, COL6A2, and COL6A3.ResultsAmong this cohort, we identified four pathogenic heterozygous in-frame exon skipping (∆) defects in COL12A1, clustering to the thrombospondin N-terminal region and the adjacent collagenous domain (Δ52, Δ53, Δ54, and Δ56 respectively), one heterozygous COL12A1 arginine-to-cysteine substitution of unclear significance (p.(Arg1863Cys)), and compound heterozygous pathogenic COL6A1 variants (c.[98–6G>A];[301C>T]) in one proband. Variant-specific intracellular accumulation of collagen XII chains, extracellular overmodification of the long isoform and near-absence of the short isoform of collagen XII, and extracellular decrease of decorin and tenascin-X were observed for the COL12A1 variants. In contrast, the COL6A1 variants abolished collagen VI and V deposition and increased tenascin-X levels.ConclusionOur data further support the significant clinical overlap between myopathic EDS and collagen VI–related myopathies, and emphasize the variant-specific consequences of collagen XII defects.     

Phenol Red Inhibits Chondrogenic Differentiation and Affects Osteogenic Differentiation of Human Mesenchymal Stem Cells in Vitro

The purpose with this study was to investigate the effect of phenol red (PR) on chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). hMSCs were differentiated into chondrogenic and osteogenic directions in DMEM with and without PR for 2, 7, 14, 21, and 28 days. Gene expression of chondrogenic and osteogenic markers were analyzed by RT-qPCR. The presence of proteoglycans was visualized histologically. Osteogenic matrix deposition and mineralization were examined measuring the alkaline phophatase activity and calcium deposition. During chondrogenic differentiation PR decreased sox9, collagen type 2, aggrecan on day 14 and 21 (P?<?0.05), and proteoglycan synthesis on day 21 and 28. Collagen type 10 was decreased on day 21 (P?<?0.05). During osteogenic differentiation PR increased alkaline phosphatase on day 7 while decreased on day 21 (P?<?0.05). PR increased collagen type 1 on day 7, 14, and day 21 (P?<?0.05). The alkaline phosphatase activity was increased after 2, 7, and 14 days (P?<?0.05). The deposition of calcium was decreased on day 21 (P?<?0.05). Our results indicate that PR should be removed from the culture media when differentiating hMSCs into chondrogenic and osteogenic directions due to the effects on these differentiation pathways.     

Chondrogenesis of hMSC in affinity-bound TGF-beta scaffolds

Herein we describe a bio-inspired, affinity binding alginate-sulfate scaffold, designed for the presentation and sustained release of transforming growth factor beta 1 (TGF-β1), and examine its effects on the chondrogenesis of human mesenchymal stem cells (hMSCs). When attached to matrix via affinity interactions with alginate sulfate, TGF-β1 loading was significantly greater and its initial release from the scaffold was attenuated compared to its burst release (>90%) from scaffolds lacking alginate-sulfate. The sustained TGF-β1 release was further supported by the prolonged activation (14 d) of Smad-dependent (Smad2) and Smad-independent (ERK1/2) signaling pathways in the seeded hMSCs. Such presentation of TGF-β1 led to hMSC chondrogenic differentiation; differentiated chondrocytes with deposited collagen type II were seen within three weeks of in vitro hMSC seeding. By contrast, in scaffolds lacking alginate-sulfate, the effect of TGF-β1 was short-term and hMSCs could not reach a similar differentiation degree. When hMSC constructs were subcutaneously implanted in nude mice, chondrocytes with deposited type II collagen and aggrecan typical of the articular cartilage were found in the TGF-β1 affinity-bound constructs. Our results highlight the fundamental importance of appropriate factor presentation to its biological activity, namely - inducing efficient stem cell differentiation.     

Longitudinal growth curves for children with classical osteogenesis imperfecta (types III and IV) caused by structural pathogenic variants in type I collagen

PurposeGrowth deficiency is a cardinal feature of osteogenesis imperfecta (OI) types III and IV, caused by pathogenic variants in type I collagen. OI-specific longitudinal growth charts are needed for patient care.MethodsWe compiled longitudinal length, weight, head circumference, and body mass index (BMI) data from 100 children with types III and IV OI and known type I collagen pathogenic variants. Effects of gender, OI type, and pathogenic variant were examined using multilevel modeling. OI-specific centile curves were constructed using generalized additive model for location, scale, and shape (GAMLSS).ResultsOI type and gender, but not the specific mutated collagen gene, significantly affect stature, but only OI type affects weight. Head circumference was not significantly different by gender, type, or mutated gene. In both genders, length curves for types III and IV OI overlap and the type IV 95th centile curve overlaps the lower US Centers for Disease Control and Prevention (CDC) curves for the general population. A pubertal growth spurt is generally absent or blunted in types III/IV OI. The body mass index 50th and 95th centile curves are distinctly shifted above respective US CDC curves in both genders.ConclusionsOI type is a stronger contributing factor than gender for OI growth, while curves do not differ for COL1A1 versus COL1A2 pathogenic variants. Types III and IV OI-specific growth curves are presented.