Mesenchymal Condensation Research Articles

Osteogenic and Angiogenic Profiles of Mandibular Bone-Forming Cells.

The mandible is a tooth-bearing structure involving one of the most prominent bones of the facial region. Mesenchymal cell condensation is the first morphological sign of osteogenesis, and several studies have focused on this stage also in the mandible. Little information is available about the early post-condensation period, during which avascular soft condensation turns into vascularized bone, and all three major bone cell types, osteoblasts, osteocytes, and osteoclasts, differentiate. In the mouse first lower molar region, the post-condensation period corresponds to the prenatal days 13–15. If during this critical period, when osteogenesis reaches the point of major bone cell differentiation, vascularization already has to play a critical role, one should be able to show molecular changes which support both types of cellular events. The aim of the present report was to follow in organ context the expression of major osteogenic and angiogenic markers and identify those that are up- or downregulated during this period. To this end, PCR Array was applied covering molecules involved in osteoblastic cell proliferation, commitment or differentiation, extracellular matrix (ECM) deposition, mineralisation, osteocyte maturation, angiogenesis, osteoclastic differentiation, and initial bone remodeling. From 161 analyzed osteogenic and angiogenic factors, the expression of 37 was altered when comparing the condensation stage with the bone stage. The results presented here provide a molecular survey of the early post-condensation stage of mandibular/alveolar bone development which has not yet been investigated in vivo.

Fibroblast growth factors in skeletal development.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are expressed throughout all stages of skeletal development. In the limb bud and in cranial mesenchyme, FGF signaling is important for formation of mesenchymal condensations that give rise to bone. Once skeletal elements are initiated and patterned, FGFs regulate both endochondral and intramembranous ossification programs. In this chapter, we review functions of the FGF signaling pathway during these critical stages of skeletogenesis, and explore skeletal malformations in humans that are caused by mutations in FGF signaling molecules.

Region-specific endodermal signals direct neural crest cells to form the three middle ear ossicles.

Defects in the middle ear ossicles - malleus, incus and stapes - can lead to conductive hearing loss. During development, neural crest cells (NCCs) migrate from the dorsal hindbrain to specific locations in pharyngeal arch (PA) 1 and 2, to form the malleus-incus and stapes, respectively. It is unclear how migratory NCCs reach their proper destination in the PA and initiate mesenchymal condensation to form specific ossicles. We show that secreted molecules sonic hedgehog (SHH) and bone morphogenetic protein 4 (BMP4) emanating from the pharyngeal endoderm are important in instructing region-specific NCC condensation to form malleus-incus and stapes, respectively, in mouse. Tissue-specific knockout of Shh in the pharyngeal endoderm or Smo (a transducer of SHH signaling) in NCCs causes the loss of malleus-incus condensation in PA1 but only affects the maintenance of stapes condensation in PA2. By contrast, knockout of Bmp4 in the pharyngeal endoderm or Smad4 (a transducer of TGFβ/BMP signaling) in the NCCs disrupts NCC migration into the stapes region in PA2, affecting stapes formation. These results indicate that region-specific endodermal signals direct formation of specific middle ear ossicles.

Mesenchymal Cells Invasion During Initial Stages of Chondrogenesis

Mesenchymal cell invasion contributes to cartilage growth. The current study investigates mesenchymal cells during the initial stages of cartilage development. Mesenchymal cell invasion was detected during stages of mesenchymal condensation, the cartilage primordia upon differentiation of the pre-chondrocytes as well as fully differentiated chondrocytes. Mesenchymal cells located at multiple sites in cartilage primordia of wide surface area with the progression of age. The mesenchymal invasion was identified in cartilage primordia of the prospective body of the basioccipital bone, basipterygoid processes of the basioccipital bone, the optic process of the quadrate bone and cartilage primordia of the dentary, frontal bone, and the sternum. In conclusion, the invasive phenomenon of mesenchymal cells is not only limited to cartilage growth but also associated with early cartilage development.

Sphenoid bone hypoplasia is a skeletal phenotype of cleidocranial dysplasia in a mouse model and patients.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder caused by heterozygous mutations in RUNX2. Affected individuals exhibit delayed maturation or hypoplasia in various bones, mainly including those formed by intramembranous ossification. Although several reports described deformation of the sphenoid bone in CCD patients, details of the associated changes have not been well documented. Most parts of the sphenoid bone are formed by endochondral ossification; however, the medial pterygoid process is formed by intramembranous ossification associated with secondary cartilage. We first investigated histological changes in the medial pterygoid process during different developmental stages in Runx2+/+ and Runx2+/- mice, finding that mesenchymal cell condensation of the anlage of this structure was delayed in Runx2+/- mice as compared with that in Runx2+/+ mice. Additionally, in Runx2+/+ mice, Osterix-positive osteoblastic cells appeared at the upper region of the anlage of the medial pterygoid process, and bone trabeculae appeared to associate with subsequent secondary cartilage formation. By contrast, few Osterix-positive osteoblastic cells appeared at the upper region of the anlage of the medial pterygoid process, and no bone trabeculae appeared thereafter in Runx2+/- mice. At more advanced embryonic stages, endochondral ossification occurred at the lower part of the medial pterygoid process in both Runx2+/+ and Runx2+/- mice. After birth, well-developed bone trabeculae occupied two-thirds of the cranial side of the medial pterygoid process, and cartilage appeared beneath these bones in Runx2+/+ mice, whereas thin trabecular bone appeared at the center of the cartilage of the medial pterygoid process in Runx2+/- mice. In adult mice, the body and medial pterygoid processes of the sphenoid bone comprised mature bones in both Runx2+/+ and Runx2+/- mice, although the axial length of the medial pterygoid processes was apparently lower in Runx2+/-mice as compared with that in Runx2+/+mice based on histological and micro-computed tomography (CT) examinations. Moreover, medical-CT examination revealed that in CCD patients, the medial pterygoid process of sphenoid bone was significantly shorter relative to that in healthy young adults. These results demonstrated that the medial pterygoid process of the sphenoid bone specifically exhibited hypoplasia in CCD.

SFMBT2 positively regulates SOX9 and chondrocyte proliferation.

SRY‑box9(SOX9) is the master regulator of the chondrocyte phenotype, which is essential for differentiating chondrogenic mesenchymal condensations into chondrocytes, and is involved in regulating every stage of chondrocyte differentiation. SOX9 deletion in chondrocytes at the late stages of cartilage development results in decreased chondrocyte proliferation; inhibited expression of cartilage matrix genes, including Indian hedgehog and the downstream parathyroid hormone‑related protein; and premature conversion of proliferating chondrocytes into hypertrophic chondrocytes, which mineralize their matrix prematurely. Therefore, SOX9 is considered vital for the majority of phases of chondrocyte lineage, from early condensations to the differentiation of proliferating chondrocytes, leading to chondrocyte hypertrophy. It has been reported that SOX9 expression is decreased in osteoarthritis(OA) cartilage. Regeneration or repair of cartilage degradation in OA remains a challenge. Previous studies have indicated that overexpression of SOX9 can promote cartilage repair and can be used as a potential therapeutic agentat the early stages of human OA. The present study identified Scm‑like with four malignant brain tumor domains2(SFMBT2) as a novel regulator of SOX9 expression in human chondrocytes. Our previous study revealed that SFMBT2 is negatively regulated in OA cartilage, and decreased levels of SFMBT2 contribute to the catabolic phenotype of chondrocytes. The present study detected increased expression levels of SFMBT2 in early cartilage development and during the early phases of chondrogenesis. Overexpression of SFMBT2 in C28/I2 cells upregulated SOX9 expression in a dose‑dependent manner. Furthermore, SFMBT2 positively regulated C28/I2 cell proliferation and restored the decreased levels of SOX9 in chondrocytes following tumor necrosis factor‑α treatment. Additional studies may reveal novel insights into the molecular mechanism involved and the potential role of SFMBT2 in cartilage repair and OA management.

Global analysis of tissue-differential gene expression patterns and functional regulation of rapid antler growth

Deer antlers are unique mammalian appendages that grow faster than any other known organ. During the rapid growth stage at 60 days after casting the previous antler, the maximal antler growth rate is up to or even more than 2 cm/day. Antler growth is driven by the growth center located in the antler tip. The growth center consists of several tissue layers from distal to proximal, including the skin, mesenchyme, precartilage, and cartilage. To analyze the gene expression patterns of the antler growth center in a tissue-differential manner and explore the molecular mechanism responsible for rapid antler growth, we used an RNA-Seq method to analyze gene expression patterns in different tissues of the Sika deer antler growth center during the rapid growth stage. We demonstrated considerable diversity in the expression levels of functional genes among different tissues within the antler growth center. These tissue-differentially expressed genes included transcription factors, growth factors, and extracellular matrix proteins. We identified a series of genes that contribute to chondrogenesis from mesenchymal cell condensation to chondrocyte differentiation. The genes identified in the antler growth center at the rapid growth stage provide valuable insight into the physiological mechanisms underlying rapid antler growth.

Hair follicle dermal condensation forms via Fgf20 primed cell cycle exit, cell motility, and aggregation

Mesenchymal condensation is a critical step in organogenesis, yet the underlying molecular and cellular mechanisms remain poorly understood. The hair follicle dermal condensate is the precursor to the permanent mesenchymal unit of the hair follicle, the dermal papilla, which regulates hair cycling throughout life and bears hair inductive potential. Dermal condensate morphogenesis depends on epithelial Fibroblast Growth Factor 20 (Fgf20). Here, we combine mouse models with 3D and 4D microscopy to demonstrate that dermal condensates form de novo and via directional migration. We identify cell cycle exit and cell shape changes as early hallmarks of dermal condensate morphogenesis and find that Fgf20 primes these cellular behaviors and enhances cell motility and condensation. RNAseq profiling of immediate Fgf20 targets revealed induction of a subset of dermal condensate marker genes. Collectively, these data indicate that dermal condensation occurs via directed cell movement and that Fgf20 orchestrates the early cellular and molecular events.

Morphogenesis of the Sternum in Quail Embryos

The flat bone develops through intramembranous ossification, in which themesenchymal cells are directly driven towards osteogenic lineage without the formation of cartilage template. While long bone develops through endochondral ossification, where cartilage template act as an intermediate stage between mesenchymal and bone tissues. Although the avian sternum is a flat bone, some studies describe formation of a cartilage template during its development. The aim of the current study was to observethe mechanism of ossification in quail sternum during embryonic development. Thirty quail embryos were collected for the current study (5 embryos/ day) during the period between Day (D) 5 and D10 of embryonic development and processed for light microscopy. The differentiation of mesenchymal condensation in to the chondrogenic cells was observed at D5whereas the secretion of extracellular matrix could be evident at D6. The cartilage primordia were observed by D7 which wereconsisted of chondrocytes, embedded in matrix and surrounded by perichondrium. Later these primordia were developed in to cartilage template by D8 where the chondrocytes were present in their lacuna. This template attained the shape of future sternum byD9, which was more distinct at D10. Thesepreliminary observationssuggested thatthe quail sternum grows through endochondral ossification. The future study will further explore the histological changes of quail sternum during post-hatching development.

Translational Control of Sox9 RNA by mTORC1 Contributes to Skeletogenesis

SummaryThe mechanistic/mammalian target of rapamycin complex 1 (mTORC1) regulates cellular function in various cell types. Although the role of mTORC1 in skeletogenesis has been investigated previously, here we show a critical role of mTORC1/4E-BPs/SOX9 axis in regulating skeletogenesis through its expression in undifferentiated mesenchymal cells. Inactivation of Raptor, a component of mTORC1, in limb buds before mesenchymal condensations resulted in a marked loss of both cartilage and bone. Mechanistically, we demonstrated that mTORC1 selectively controls the RNA translation of Sox9, which harbors a 5′ terminal oligopyrimidine tract motif, via inhibition of the 4E-BPs. Indeed, introduction of Sox9 or a knockdown of 4E-BP1/2 in undifferentiated mesenchymal cells markedly rescued the deficiency of the condensation observed in Raptor-deficient mice. Furthermore, introduction of the Sox9 transgene rescued phenotypes of deficient skeletal growth in Raptor-deficient mice. These findings highlight a critical role of mTORC1 in mammalian skeletogenesis, at least in part, through translational control of Sox9 RNA.

Character definition and tempus optimum in comparative chondrocranial research

AbstractHere, we define a research program using primordial skull characters for broad scale phylogenetic comparisons, particularly for amniotes. There are many chondrocranium descriptions in the literature, encompassing many clades of vertebrates. The definition of a comparable “tempus optimum” for fully formed chondrocrania, as developed herein, will enable discrete character definition in the sense of phylogenetic systematics. The “tempus optimum” is a time period in which the nose region shows full chondrification and the basicranium has not yet changed in its anatomy by ossification processes. For studies on heterochrony, changes in the developmental timing through evolution, the available data in the literature are insufficient. Anatomical standardizations, traceable methodological approaches and clear definitions of comparable developmental stages are needed. Moreover, for profound homology assertions of chondrocranial structures, ontogeny needs to be traced from early mesenchymal condensation, through several stages of cartilage development, up to postnatal stages, in which ossifications are already present. A holistic observation, including hard and soft tissue anatomy, is of paramount importance to generate hypotheses of homology with much explanatory power and to derive conclusions on functional morphology. For that, traditional histological methodology is needed. We exemplify the suggested approach in two case studies on the disputed laterosphenoid in turtles and crocodiles and on the homology of pila antotica in a pleurodire turtle, and, using here defined criteria, we formulate hypotheses on their homology.

Tracing the developmental origin of a lizard skull: Chondrocranial architecture, heterochrony, and variation in lacertids.

The sand lizard, Lacerta agilis, is a classical model species in herpetology. Its adult skull anatomy and its embryonic development are well known. The description of its fully formed primordial skull by Ernst Gaupp, in 1900, was a key publication in vertebrate morphology and influenced many comparative embryologists. Based on recent methodological considerations, we restudied the early cranial development of this species starting as early as the formation of mesenchymal condensations up to the fully formed chondrocranium. We traced the formation of the complex chondrocranial architecture in detail, clarified specific homologies for the first time, and uncovered major differences to old textbook descriptions. Comparison with other lacertid lizards revealed a very similar genesis of the primordial skull. However, we detected shifts in the developmental timing of particular cartilaginous elements, mainly in the nasal region, which may correlate to specific ecological adaptation in the adults. Late timing of nasal elements might be an important innovation for the successful wide range distribution of the well-known sand lizard.

Correlating interfacial water dynamics with protein-protein interaction in complex of GDF-5 and BMPRI receptors

GDF-5 mediated signal transduction regulating chondrogenesis and skeletogenesis involves three different type-I receptors viz. Act-RI, BMPRIA and BMPRIB. BMPRIA and BMPRIB generally shows temporal and spatial co-expression but some spatially different expression pattern has also been observed. BMPRIA receptor is the key receptor implicated in BMP signalling during osteogenesis and is expressed in osteoblasts during the course of bone formation. However, BMPRIB appears to be primarily expressed in mesenchymal pre-cartilage condensations and also found in differentiated osteoblast and chondrocytes. The extracellular pH affects bone cell function and it is experimentally known that mineralization of bone is affected by shift of pH in cultured osteoblast. Here we report the effect of pH on dynamics of water present at the interface of GDF-5:BMPRIA and GDF-5:BMPRIB and binding interaction energy of these complexes. Water dynamics at different pH was analysed using residence time and hydrogen bond relaxation kinetics. pH influences the interaction energy between GDF-5 and BMPRIA and BMPRIB receptors indicating the electrostatic environment modulating the activity of two receptors. This pH dependence of interaction energy is further supported by similar behaviour of hydrogen bond existence of buried water molecules at the interface. In contrast to this the slow and fast exchanging water molecules do not show similar pH dependence of hydrogen bonding relaxation kinetics. Hence; we conclude that only buried water molecule at the interface influences the protein-protein interaction and the electrostatic environment of the extracellular fluid might decide the specificity of the two receptors.

Formirovanie pozvonochnogo kanala v ehmbriogeneze cheloveka

In the study, 29 totally cleared and double-stained human embryos of 7-12 weeks of gestational age and 33series of histological sections of 7-70 mm CRL human embryos were used. At the 5th week of gestation, the vertebrae are presented by mesenchymal condensations around the notochord. In 7 week embryos, separate centers of chondrification are identified in the vertebral primordia. At the end of the embryonic period (8th week of development) due to the fusion of the centers of chondrification, the vertebral primordia are presented by whole cartilaginous structures having a centrum located in front of the spinal cord and two neural processes that span it from two sides. The body of the future vertebrae is located on the ventral side of the spinal cord, the anteroposterior size of which is approximately twice as large as the similar size of the cartilaginous primordium of vertebral body. The neural processes are directed dorsally and cover only 3/4 of the circumference of the spinal cord from both sides. As a result, the vertebral foramen remains open behind and there is no spinous process. The neural arches of all thoracic and upper lumbar vertebrae fuse in fetuses of the 10th week of gestation. At the 10th week of gestation, the centers of ossification appear in the neural processes of C2-Th8. The primary centers of ossification in the ribs appear from the 8th week of embryogenesis. It is established that the fusion of the neural processes behind the spinal cord begins with the lower cervical - upper thoracic vertebrae and then the process spreads in the cranial and caudal directions. Simultaneously with the fusion of the neural processes, a spinous process of the vertebrae is formed. The formation of the spinal canal is completed in 12-week human fetuses. At all stages of development of the vertebral primordia, their close interaction with the notochord is traced. Notochord plays an important role in the morphogenesis of axial structures. The congenital neural tube defect (anencephaly) is combined with the spina bifida and fusion of adjacent vertebral arches.

Notum attenuates Wnt/β–catenin signaling to promote tracheal cartilage patterning

Tracheobronchomalacia (TBM) is a common congenital disorder in which the cartilaginous rings of the trachea are weakened or missing. Despite the high prevalence and clinical issues associated with TBM, the etiology is largely unknown. Our previous studies demonstrated that Wntless (Wls) and its associated Wnt pathways are critical for patterning of the upper airways. Deletion of Wls in respiratory endoderm caused TBM and ectopic trachealis muscle. To understand mechanisms by which Wls mediates tracheal patterning, we performed RNA sequencing in prechondrogenic tracheal tissue of Wlsf/f;ShhCre/wt embryos. Chondrogenic Bmp4, and Sox9 were decreased, while expression of myogenic genes was increased. We identified Notum, a deacylase that inactivates Wnt ligands, as a target of Wls induced Wnt signaling. Notum’s mesenchymal ventral expression in prechondrogenic trachea overlaps with expression of Axin2, a Wnt/β-catenin target and inhibitor. Notum is induced by Wnt/β-catenin in developing trachea. Deletion of Notum activated mesenchymal Wnt/β-catenin and caused tracheal mispatterning of trachealis muscle and cartilage as well as tracheal stenosis. Notum is required for tracheal morphogenesis, influencing mesenchymal condensations critical for patterning of tracheal cartilage and muscle. We propose that Notum influences mesenchymal cell differentiation by generating a barrier for Wnt ligands produced and secreted by airway epithelial cells to attenuate Wnt signaling.

Swarming Migration of Co-attracting Mesenchymal Cells into Fractal-Like Epithelial Clusters

Cell-secreted factors can function as biochemical “signal relays” to coordinate collective migration over long distances. For instance, cells “co-attract” by releasing chemokines that they also chemotax towards, driving aggregation and cohesion during neural crest development, mesenchymal condensation, and neutrophil homing. Here, we show that mammary epithelial cells cultured in reduced serum conditions display a mutual attraction that results in self-organizing migration patterns. Ordinarily, the migration of spatially dispersed cells in EGF-supplemented media can be understood as a persistent random walk. Instead, cells cultured in EGF-depleted media display directed migration towards each other through co-attraction, resulting in multicellular aggregation. Subsequently, aggregated cells establish cell-cell junctions with E-cadherin, reminiscent of a mesenchymal-to-epithelial transition (MET). Furthermore, cells exhibit substantial morphological changes, extending outward protrusions towards other cells in the proximity. As a consequence, these aggregates display a highly branched, fractal-like architecture. This biological behavior has physical analogies with diffusion-limited aggregation of colloidal particles, which results in a gelation or jamming transition even at very subconfluent cell densities. We implement a computational model based on dissipative particle dynamics that recapitulates clustering and aggregation. These results may reveal new quantitative insights into swarming cell migration during neural crest development, mesenchymal condensation, and tumor metastasis.

Dose and Timing of N‐Cadherin Mimetic Peptides Regulate MSC Chondrogenesis within Hydrogels

The transmembrane glycoprotein N-cadherin (NCad) mediates cell-cell interactions found during mesenchymal condensation and chondrogenesis. Here, NCad-derived peptides (i.e., HAV) are incorporated into hyaluronic acid (HA) hydrogels with encapsulated mesenchymal stem cells (MSCs). Since the dose and timing of NCad signaling are dynamic, HAV peptide presentation is tuned via alterations in peptide concentration and incorporation of an ADAM10-cleavable domain between the hydrogel and the HAV motif, respectively. HA hydrogels functionalized with HAV result in dose-dependent increases in early chondrogenesis of encapsulated MSCs and resultant cartilage matrix production. For example, type II collagen and glycosaminoglycan production increase ≈9- and 2-fold with the highest dose of HAV (i.e., 2 × 10-3 m), respectively, when compared to unmodified hydrogels, while incorporation of an efficient ADAM10-cleavable domain between the HAV peptide and hydrogel abolishes increases in chondrogenesis and matrix production. Treatment with a small-molecule ADAM10 inhibitor restores the functional effect of the HAV peptide, indicating that timing and duration of HAV peptide presentation is crucial for robust chondrogenesis. This study demonstrates a nuanced approach to the biofunctionalization of hydrogels to better emulate the complex cell microenvironment during embryogenesis toward stem-cell-based cartilage production.

Intracellular biosynthesis of lipids and cholesterol by Scap and Insig in mesenchymal cells regulates long bone growth and chondrocyte homeostasis.

ABSTRACTDuring enchondral ossification, mesenchymal cells express genes regulating the intracellular biosynthesis of cholesterol and lipids. Here, we have investigated conditional deletion of Scap or of Insig1 and Insig2 (Scap inhibits intracellular biosynthesis and Insig proteins activate intracellular biosynthesis). Mesenchymal condensation and chondrogenesis was disrupted in mice lacking Scap in mesenchymal progenitors, whereas mice lacking the Insig genes in mesenchymal progenitors had short limbs, but normal chondrogenesis. Mice lacking Scap in chondrocytes showed severe dwarfism, with ectopic hypertrophic cells, whereas deletion of Insig genes in chondrocytes caused a mild dwarfism and shortening of the hypertrophic zone. In vitro studies showed that intracellular cholesterol in chondrocytes can derive from exogenous and endogenous sources, but that exogenous sources cannot completely overcome the phenotypic effect of Scap deficiency. Genes encoding cholesterol biosynthetic proteins are regulated by Hedgehog (Hh) signaling, and Hh signaling is also regulated by intracellular cholesterol in chondrocytes, suggesting a feedback loop in chondrocyte differentiation. Precise regulation of intracellular biosynthesis is required for chondrocyte homeostasis and long bone growth, and these data support pharmacological modulation of cholesterol biosynthesis as a therapy for select cartilage pathologies.

Mimetic Hierarchical Approaches for Osteochondral Tissue Engineering.

Biomimetic OC TE uses "smart" scaffolds able to locally regulate cell phenotypes and dual-flow bioreactors for two sets of conditions for cartilage/bone Protocols for hierarchical OC grafts engineering should entail mesenchymal condensation for cartilage and vascular component for bone Immunomodulation, low oxygen tension, purinergic signaling, time dependence of stimuli application are important aspects to consider in biomimetic OC TE.

High-density human mesenchymal stem cell rings with spatiotemporally-controlled morphogen presentation as building blocks for engineering bone diaphyseal tissue.

Emerging biomimetic tissue engineering strategies aim to partially recapitulate fundamental events that transpire during embryonic skeletal development; namely, cellular self-organization and targeted morphogenetic pathway activation. Here, we describe self-assembled, scaffold-free human mesenchymal stem cell (hMSC) rings featuring microparticle-mediated presentation of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2). We tested the hypothesis that spatiotemporally-controlled dual presentation of TGF-β1 and BMP-2 is superior in modulating in vitro endochondral ossification of high-density cellular constructs compared to single morphogen delivery. hMSC rings were engineered by seeding cells with microparticles presenting (1) TGF-β1, (2) BMP-2, or (3) TGF-β1 + BMP-2 in custom agarose wells to facilitate self-assembly within 2 d, followed by horizontal culture on glass tubes for 5 weeks. At day 2, hMSC rings across groups revealed homogenous cellular organization mimetic of early mesenchymal condensation with no evidence of new matrix or mineral deposition. Significant early chondrogenic and osteogenic priming occurred with TGF-β1 + BMP-2 presentation compared to single morphogen-loaded groups. By week 5, TGF-β1-loaded hMSC rings had undergone chondrogenesis, while presentation of BMP-2 alone or in conjunction with TGF-β1 stimulated chondrogenesis, chondrocyte hypertrophy, and osteogenesis indicative of endochondral ossification. Importantly, tissue mineralization was most compelling with TGF-β1 + BMP-2 loading. Lastly, hMSC ring 'building blocks' were shown to efficiently fuse into tubes within 6 d post self-assembly. The resulting tubular tissue units exhibited structural integrity, highlighting the translational potential of this advanced biomimetic technology for potential early implantation in long bone defects.