3D printing of Ceffe-infused scaffolds for tailored nipple-like cartilage development.

The reconstruction of a stable, nipple-shaped cartilage graft that precisely matches the natural nipple in shape and size on the contralateral side is a clinical challenge. While 3D printing technology can efficiently and accurately manufacture customized complex structures, it faces limitations due to inadequate blood supply, which hampers the stability of nipple-shaped cartilage grafts produced using this technology. To address this issue, we employed a biodegradable biomaterial, Poly(lactic-co-glycolic acid) (PLGA), loaded with Cell-Free Fat Extract (Ceffe). Ceffe has demonstrated the ability to promote angiogenesis and cell proliferation, making it an ideal bio-ink for bioprinting precise nipple-shaped cartilage grafts. We utilized the Ceffe/PLGA scaffold to create a porous structure with a precise nipple shape. This scaffold exhibited favorable porosity and pore size, ensuring stable shape maintenance and satisfactory biomechanical properties. Importantly, it could release Ceffe in a sustained manner. Our in vitro results confirmed the scaffold's good biocompatibility and its ability to promote angiogenesis, as evidenced by supporting chondrocyte proliferation and endothelial cell migration and tube formation. Furthermore, after 8 weeks of in vivo culture, the Ceffe/PLGA scaffold seeded with chondrocytes regenerated into a cartilage support structure with a precise nipple shape. Compared to the pure PLGA group, the Ceffe/PLGA scaffold showed remarkable vascular formation, highlighting the beneficial effects of Ceffe. These findings suggest that our designed Ceffe/PLGA scaffold with a nipple shape represents a promising strategy for precise nipple-shaped cartilage regeneration, laying a foundation for subsequent nipple reconstruction.

Fermentation supernatant of Staphylococcus aureus drives catabolism in chondrocytes via NF-κB signaling mediated increase of cholesterol metabolism.

Septic arthritis induced by Staphylococcus aureus (S. aureus) causes irreversible cartilage degradation and subsequent permanent joint dysfunction. Recently, cartilage degradation in osteoarthritis is recognized to be associated with metabolic disorders. However, whether cholesterol metabolism is linked to septic arthritis pathology remains largely unknown. Here, we found that exposure to fermentation supernatant (FS) of S. aureus in chondrocytes resulted in a significant increase in expression of key modulators involved in cholesterol metabolism, including lectin-type oxidized low density lipoprotein receptor 1 (LOX1), cholesterol 25-hydroxylase (CH25H), 25- hydroxycholesterol 7α-hydroxylase (CYP7B1) as well as retinoic acid-related orphan receptor alpha (RORα), a binding receptor for cholesterol metabolites. We further demonstrated that enhancement of CH25H/CYP7B1/RORα axis resulted from FS exposure was mediated by activation of NF-κB signaling, along with upregulation in catabolic factors including matrix metallopeptidases (MMP3 and MMP13), aggrecanase-2 (ADAMTS5), and nitric oxide synthase-2 (NOS2) in chondrocytes. Exogenous cholesterol acts synergistically with FS in activating NF-κB pathway and increases cholesterol metabolism. While, the addition of tauroursodeoxycholic acid (TUDCA) which promotes cholesterol efflux, resulted in remarkable reduction of intracellular cholesterol level and restoration of balance between anabolism and catabolism in FS treated chondrocytes. Collectively, our data indicated that, in response to FS of S. aureus, NF-κB signaling activation coupled with increased cholesterol metabolism to stimulate catabolic factors in chondrocytes, highlighting cholesterol metabolism as a potential therapeutic target for treating septic arthritis.

Trabecular Bone Parameters, TIMP-2, MMP-8, MMP-13, VEGF Expression and Immunolocalization in Bone and Cartilage in Newborn Offspring Prenatally Exposed to Fumonisins.

Fumonisins are protein serine/threonine phosphatase inhibitors and potent inhibitors of sphingosine N-acyltransferase (ceramide synthase) disrupting de novo sphingolipid biosynthesis. The experiment was conducted to evaluate the effects of fumonisins (FB) exposure from the 7th day of pregnancy to parturition on offspring bone development. The rats were randomly allocated to either a control group (n = 6), not treated with FBs, or to one of the two groups intoxicated with FBs (either at 60 mg FB/kg b.w. or at 90 mg FB/kg b.w. Numerous negative, offspring sex-dependent effects of maternal FB exposure were observed with regards to the histomorphometry of trabecular bone. These effects were due to FB-inducted alterations in bone metabolism, as indicated by changes in the expression of selected proteins involved in bone development: tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8), matrix metalloproteinase 13 (MMP-13), and vascular endothelial growth factor (VEGF). The immunolocalization of MMPs and TIMP-2 was performed in trabecular and compact bone, as well as articular and growth plate cartilages. Based on the results, it can be concluded that the exposure of pregnant dams to FB negatively affected the expression of certain proteins responsible for bone matrix degradation in newborns prenatally exposed to FB in a dose- and sex-dependent manner.

M1-like macrophage contributes to chondrogenesis in vitro.

Cartilage tissues have poor self-repairing abilities. Regenerative medicine can be applied to recover cartilage tissue damage in the oral and maxillofacial regions. However, hitherto it has not been possible to predict the maturity of the tissue construction after transplantation or to prepare mature cartilage tissues before transplantation that can meet clinical needs. Macrophages play an important role in cartilage tissue regeneration, although the exact mechanisms remain unknown. In this study, we established and verified an in vitro experimental system for the direct co-culture of cell pellets prepared from mouse auricular chondrocytes and macrophages polarized into four phenotypes (M1-like, M1, M2-like, and M2). We demonstrate that cartilage pellets co-cultured with M1-like promoted collagen type 2 and aggrecan production and induced the most significant increase in chondrogenesis. Furthermore, M1-like shifted to M2 on day 7 of co-culture, suggesting that the cartilage pellet supplied factors that changed the polarization of M1-like. Our findings suggest that cartilage regenerative medicine will be most effective if the maturation of cartilage tissues is induced in vitro by co-culture with M1-like before transplantation.

Fabrication of Robust, Shape Recoverable, Macroporous Bacterial Cellulose Scaffolds for Cartilage Tissue Engineering.

Recently, the fabricating of three-dimensional (3D) macroporous bacterial cellulose (MP-BC) scaffolds with mechanically disintegrated BC fragments has attracted considerable attention. However, the successful implementation of these materials depends mainly on their mechanical stability and robustness. Here, a non-toxic crosslinker, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), is employed to induce crosslinking reactions between BC fragments. In addition to their large pore sizes, the EDC/NHS-crosslinked MP-BC scaffolds exhibit excellent compression properties and shape recovery ability, owing to the EDC/NHS-induced crosslinking on the BC nanofibers. The results of in vitro studies reveal that the biocompatibility of MP-BC scaffolds is better than that of pristine BC scaffolds because the former provided more space for cell proliferation. The results of in vivo studies show that the neocartilage tissue with native cartilage appearance and abundant cartilage-specific extracellular matrix deposition is successfully regenerated in nude mice. The findings reveal the immense application potential of mechanically robust BC scaffolds with controllable pore sizes and shape-recoverable properties in tissue engineering.

Cell Sources for Cartilage Repair-Biological and Clinical Perspective.

Cell-based therapy represents a promising treatment strategy for cartilage defects. Alone or in combination with scaffolds/biological signals, these strategies open many new avenues for cartilage tissue engineering. However, the choice of the optimal cell source is not that straightforward. Currently, various types of differentiated cells (articular and nasal chondrocytes) and stem cells (mesenchymal stem cells, induced pluripotent stem cells) are being researched to objectively assess their merits and disadvantages with respect to the ability to repair damaged articular cartilage. In this paper, we focus on the different cell types used in cartilage treatment, first from a biological scientist's perspective and then from a clinician's standpoint. We compare and analyze the advantages and disadvantages of these cell types and offer a potential outlook for future research and clinical application.

Characterization of the growth plate-bone interphase region using cryo-FIB SEM 3D volume imaging.

The interphase region at the base of the growth plate includes blood vessels, cells and mineralized tissues. In this region, cartilage is mineralized and replaced with bone. Blood vessel extremities permeate this space providing nutrients, oxygen and signaling factors. All these different components form a complex intertwined 3D structure. Here we use cryo-FIB SEM to elaborate this 3D structure without removing the water. As it is challenging to image mineralized and unmineralized tissues in a hydrated state, we provide technical details of the parameters used. We obtained two FIB SEM image stacks that show that the blood vessels are in intimate contact not only with cells, but in some locations also with mineralized tissues. There are abundant red blood cells at the extremities of the vessels. We also documented large multinucleated cells in contact with mineralized cartilage and possibly also with bone. We observed membrane bound mineralized particles in these cells, as well as in blood serum, but not in the hypertrophic chondrocytes. We confirm that there is an open pathway from the blood vessel extremities to the mineralizing cartilage. Based on the sparsity of the mineralized particles, we conclude that mainly ions in solution are used for mineralizing cartilage and bone, but these are augmented by the supply of mineralized particles.

Alterations in cartilage quantification before and after injections of mesenchymal stem cells into osteoarthritic knees.

Several studies have reported improvement in knee pain following mesenchymal stem cell (MSC) injections for knee osteoarthritis (OA). We developed a novel 3D magnetic resonance imaging (MRI) analysis software program that provides "projected cartilage area ratios" for automatic detection of changes in cartilage amounts. The primary objective of this prospective interventional study was to compare alterations in the projected cartilage area ratio (thickness ≥ 1.5 mm) at the femoral posteromedial region between 30 weeks before and 30 weeks after synovial MSC injections. Secondary objectives were to assess the clinical scores and safety of MSC injections. Patients with OA who complained of knee pain underwent autologous synovial MSC injections into the knee at time 0 and again 15 weeks later. MRI examinations were performed at - 30, - 15, - 1, and 30 weeks. Patients showing < 3% decreases in the projected cartilage area ratio (thickness ≥ 1.5 mm) at the femoral the posteromedial region from - 30 weeks to - 15 weeks were excluded from the study. The Lysholm Knee Score, Knee Injury and Osteoarthritis Outcome Scale (KOOS), and Numerical Rating Scale (NRS) scores were evaluated at - 30, - 15, - 5, - 2, 0, 5, 10, 15, 20, 25, and 30 weeks. Five patients were excluded because 3D MRI analysis showed no cartilage loss at - 15 weeks. Ultimately, eight OA patients underwent MSC injections. The projected cartilage area ratio significantly decreased by 0.07 in the 30 weeks before MSC injections (p = 0.01), but no further decreases occurred in the 30 weeks after MSC injections. The projected cartilage area ratio at the femoral posteromedial region showed a significant difference between 30 weeks before and 30 weeks after MSC injections. The Lysholm Knee Score, KOOS, and NRS values improved significantly after the injections. MSC injection could not be ruled out as the cause of two adverse events: transient knee pain and itching in both hands. Fully automatic 3D MRI analysis showed that synovial MSC injections suppressed cartilage loss in patients with progressive OA.Trial registration: Intraarticular injections of synovial stem cells for osteoarthritis of the knee (Number UMIN 000026732). Date of registration; June 1, 2017. https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000029967 .

Mammalian tracheal development and reconstruction: insights from in vivo and in vitro studies.

The trachea delivers inhaled air into the lungs for gas exchange. Anomalies in tracheal development can result in life-threatening malformations, such as tracheoesophageal fistula and tracheomalacia. Given the limitations of current therapeutic approaches, development of technologies for the reconstitution of a three-dimensional trachea from stem cells is urgently required. Recently, single-cell sequencing technologies and quantitative analyses from cell to tissue scale have been employed to decipher the cellular basis of tracheal morphogenesis. In this Review, recent advances in mammalian tracheal development and the generation of tracheal tissues from pluripotent stem cells are summarized.

Intrinsic and growth-mediated cell and matrix specialization during murine meniscus tissue assembly.

The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1, and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences in meniscus zones were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.

The utility of biomedical scaffolds laden with spheroids in various tissue engineering applications.

A spheroid is a complex, spherical cellular aggregate supporting cell-cell and cell-matrix interactions in an environment that mimics the real-world situation. In terms of tissue engineering, spheroids are important building blocks that replace two-dimensional cell cultures. Spheroids replicate tissue physiological activities. The use of spheroids with/without scaffolds yields structures that engage in desired activities and replicate the complicated geometry of three-dimensional tissues. In this mini-review, we describe conventional and novel methods by which scaffold-free and scaffolded spheroids may be fabricated and discuss their applications in tissue regeneration and future perspectives.

Spatiotemporal Immunomodulation Using Biomimetic Scaffold Promotes Endochondral Ossification-Mediated Bone Healing.

Biomaterials play an important role in treating bone defects by promoting direct osteogenic healing through intramembranous ossification (IO). However, majority of the body's bones form via cartilaginous intermediates by endochondral ossification (EO), a process that has not been well mimicked by engineered scaffolds, thus limiting their clinical utility in treating large segmental bone defects. Here, by entrapping corticosteroid dexamethasone within biomimetic recombinant human bone morphogenetic protein (rhBMP)-loaded porous mesoporous bioglass scaffolds and regulating their release kinetics, significant degree of ectopic bone formation through endochondral ossification is achieved. By regulating the recruitment and polarization of immune suppressive macrophage phenotypes, the scaffold promotes rapid chondrogenesis by activating Hif-3α signaling pathway in mesenchymal stem cells, which upregulates the expression of downstream chondrogenic genes. Inhibition of Hif-3α signaling reverses the endochondral ossification phenotype. Together, these results reveal a strategy to facilitate developmental bone growth process using immune modulating biomimetic scaffolds, thus providing new opportunities for developing biomaterials capable of inducing natural tissue regeneration.

Endogenous FGF-2 levels impact FGF-2/BMP-2 growth factor delivery dosing in aged murine calvarial bone defects.

Bone repair in elderly mice has been shown to be improved or negatively impacted by supplementing the highly osteogenic bone morphogenetic protein-2 (BMP-2) with fibroblast growth factor-2 (FGF-2). To better predict the outcome of FGF-2 supplementation, we investigated whether endogenous levels of FGF-2 play a role in optimal dosing of FGF-2 for augmenting BMP-2 activity in elderly mice. In vivo calvarial bone defect studies in Fgf2 knockout mice with wildtype controls were conducted with the growth factors delivered in a highly localized manner from a biomimetic calcium phosphate/polyelectrolyte multilayer coating applied to a bone graft substitute. Endogenous FGF-2 levels were measured in old mice versus young and found to decrease with age. Optimal dosing for improving bone defect repair correlated with levels of endogenous FGF-2, with a larger dose of FGF-2 required to have a positive effect on bone healing in the Fgf2 knockout mice. The same dose in wildtype old mice, with higher levels of FGF-2, promoted chondrogenesis and increased osteoclast activity. The results suggest a personalized medicine approach, based on a knowledge of endogenous levels of FGF-2, should guide FGF-2 supplementation in order to avoid provoking excessive bone resorption and cartilage formation, both of which inhibited calvarial bone repair.

High-Strength, Strongly Bonded Nanocomposite Hydrogels for Cartilage Repair.

Polyacrylamide-based hydrogels are widely used as potential candidates for cartilage replacement. However, their bioapplicability is sternly hampered due to their limited mechanical strength and puncture resistance. In the present work, the strength of polyacrylamide (PAM) hydrogels was increased using titanium oxide (TiO2) and carbon nanotubes (CNTs) separately and a combination of TiO2 with CNTs in a PAM matrix, which was interlinked by the bonding between nanoparticles and polymers with the deployment of density functional theory (DFT) approach. The synergistic effect and strong interfacial bonding of TiO2 and CNT nanoparticles with PAM are attributed to high compressive strength, elastic modulus (>0.43 and 2.340 MPa, respectively), and puncture resistance (estimated using the needle insertion test) for the PAM-TiO2-CNT hydrogel. The PAM-TiO2-CNT composite hydrogel revealed a significant self-healing phenomenon along with a sign toward the bioactivity and cytocompatibility by forming the apatite crystals in simulated body fluid as well as showing a cell viability of ∼99%, respectively. Furthermore, for new insights on interfacial bonding and structural and electronic features involved in the hydrogels, DFT was used. The PAM-TiO2-CNT composite model, constructed by two interfaces (PAM-TiO2 and PAM-CNT), was stabilized by H-bonding and van der Waals-type interactions. Employing the NCI plot, HOMO-LUMO gap, and natural population analysis tools, the PAM-TiO2-CNT composite has been found to be most stable. Therefore, the prepared polyacrylamide hydrogels in combination with the TiO2 and CNT can be a remarkable nanocomposite hydrogel for cartilage repair applications.

The hypertrophic cartilage induction influences the building-block capacity of human adipose stem/stromal cell spheroids for biofabrication.

As an alternative to the classical tissue engineering approach, bottom-up tissue engineering emerges using building blocks in bioassembly technologies. Spheroids can be used as building blocks to reach a highly complex ordered tissue by their fusion (bioassembly), representing the foundation of biofabrication. In this study, we analyzed the biomechanical properties and the fusion capacity of human adipose stem/stromal cell (ASC) we spheroids during an in vitro model of hypertrophic cartilage established by our research group. Hypertrophic induced-ASC spheroids showed a statistically significant higher Young's modulus at weeks 2 (P < .001) and 3 (P < .0005) compared with non-induced. After fusion, non-induced and induced-ASC spheroids increased the contact area and decreased their pairs' total length. At weeks 3 and 5, induced-ASC spheroids did not fuse completely, and the cells migrate preferentially in the fusion contact region. Alizarin red O staining showed the highest intensity of staining in the fused induced-ASC spheroids at week 5, together with intense staining for collagen type I and osteocalcin. Transmission electron microscopy and element content analysis (X-ray Energy Dispersive Spectroscopy) revealed in the fused quartet at week 3 a crystal-like structure. Hypertrophic induction interferes with the intrinsic capacity of spheroids to fuse. The measurements of contact between spheroids during the fusion process, together with the change in viscoelastic profile to the plastic, will impact the establishment of bioassembly protocols using hypertrophic induced-ASC spheroids as building blocks in biofabrication.

Smart scaffolds: shape memory polymers (SMPs) in tissue engineering.

Smart scaffolds based on shape memory polymer (SMPs) have been increasingly studied in tissue engineering. The unique shape actuating ability of SMP scaffolds has been utilized to improve delivery and/or tissue defect filling. In this regard, these scaffolds may be self-deploying, self-expanding, or self-fitting. Smart scaffolds are generally thermoresponsive or hydroresponsive wherein shape recovery is driven by an increase in temperature or by hydration, respectively. Most smart scaffolds have been directed towards regenerating bone, cartilage, and cardiovascular tissues. A vast variety of smart scaffolds can be prepared with properties targeted for a specific tissue application. This breadth of smart scaffolds stems from the variety of compositions employed as well as the numerous methods used to fabricated scaffolds with the desired morphology. Smart scaffold compositions span across several distinct classes of SMPs, affording further tunability of properties using numerous approaches. Specifically, these SMPs include those based on physically cross-linked and chemically cross-linked networks and include widely studied shape memory polyurethanes (SMPUs). Various additives, ranging from nanoparticles to biologicals, have also been included to impart unique functionality to smart scaffolds. Thus, given their unique functionality and breadth of tunable properties, smart scaffolds have tremendous potential in tissue engineering.

Phosphatase inhibition by LB-100 enhances BMN-111 stimulation of bone growth.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP in chondrocytes and severe short stature, causing achondroplasia (ACH) and acromesomelic dysplasia, type Maroteaux, respectively. Previously, we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+). Here, because FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein, we tested whether a phosphatase inhibitor (LB-100) could enhance BMN-111-stimulated bone growth in ACH. Measurements of cGMP production in chondrocytes of living tibias, and of NPR2 phosphorylation in primary chondrocytes, showed that LB-100 counteracted FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, the combination of BMN-111 and LB-100 increased bone length and cartilage area, restored chondrocyte terminal differentiation, and increased the proliferative growth plate area, more than BMN-111 alone. The combination treatment also reduced the abnormal elevation of MAP kinase activity in the growth plate of Fgfr3Y367C/+ mice and improved the skull base anomalies. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.

Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.

Decellularized Cartilage Extracellular Matrix Incorporated Silk Fibroin Hybrid Scaffolds for Endochondral Ossification Mediated Bone Regeneration.

Tissue engineering strategies promote bone regeneration for large bone defects by stimulating the osteogenesis route via intramembranous ossification in engineered grafts, which upon implantation are frequently constrained by insufficient integration and functional anastomosis of vasculature from the host tissue. In this study, we developed a hybrid biomaterial incorporating decellularized cartilage extracellular matrix (CD-ECM) as a template and silk fibroin (SF) as a carrier to assess the bone regeneration capacity of bone marrow-derived mesenchymal stem cells (hBMSC's) via the endochondral ossification (ECO) route. hBMSC's were primed two weeks for chondrogenesis, followed by six weeks for hypertrophy onto hybrid CD-ECM/SF or SF alone scaffolds and evaluated for the mineralized matrix formation in vitro. Calcium deposition biochemically determined increased significantly from 4-8 weeks in both SF and CD-ECM/SF constructs, and retention of sGAG's were observed only in CD-ECM/SF constructs. SEM/EDX revealed calcium and phosphate crystal localization by hBMSC's under all conditions. Compressive modulus reached a maximum of 40 KPa after eight weeks of hypertrophic induction. µCT scanning at eight weeks indicated a cloud of denser minerals in groups after hypertrophic induction in CD-ECM/SF constructs than SF constructs. Gene expression by RT-qPCR revealed that hBMSC's expressed hypertrophic markers VEGF, COL10, RUNX2, but the absence of early hypertrophic marker ChM1 and later hypertrophic marker TSBS1 and the presence of osteogenic markers ALPL, IBSP, OSX under all conditions. Our data indicate a new method to prime hBMSC'S into the late hypertrophic stage in vitro in mechanically stable constructs for ECO-mediated bone tissue regeneration.