Perichondrial Cells Research Articles

Bone development by Hedgehog and Wnt signaling, Runx2, and Sp7.

Hedgehog and canonical Wnt signaling pathways and the transcription factors Runx2 and Sp7 are essential for osteoblast differentiation. Ihh is necessary for the commitment of perichondrial mesenchymal cells to Runx2+ osteoprogenitors and for the formation of the bone collar and primary spongiosa. Runx2 is needed for osteoblast differentiation during both endochondral and intramembranous ossification. It regulates the commitment of mesenchymal cells to osteoblast-lineage cells and their proliferation by inducing the expression of Hedgehog, Fgf, Wnt, Pthlh signaling pathway genes, and Dlx5. The Runx2-induced expression of Fgfr2 and Fgfr3 is important for the proliferation of osteoblast-lineage cells. Runx2 induces Sp7 expression and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Runx2, Sp7, and canonical Wnt signaling induce the differentiation of preosteoblasts into osteoblasts. Canonical Wnt signaling, but not Sp7, enhances the proliferation of osteoblast-lineage cells. In mature osteoblasts, Runx2 plays an important role in the expression of major bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and Bglap/Bglap2. The canonical Wnt signaling pathway is also crucial for bone formation by mature osteoblasts. Sp7 is needed for osteocytes to acquire a sufficient number of processes and a reduction in these processes results in osteocyte apoptosis and cortical porosity.

Altered Sox9 and FGF signaling gene expression in Aga2 OI mice negatively affects linear growth.

Osteogenesis imperfecta (OI), or brittle bone disease, is a disorder characterized by bone fragility and increased fracture incidence. All forms of OI also feature short stature, implying an effect on endochondral ossification. Using the Aga2+/- mouse, which has a mutation in type I collagen, we show an affected growth plate primarily due to a shortened proliferative zone. We used single-cell RNA-Seq analysis of tibial and femoral growth plate tissues to understand transcriptional consequences on growth plate cell types. We show that perichondrial cells, which express abundant type I procollagen, and growth plate chondrocytes, which were found to express low amounts of type I procollagen, had ER stress and dysregulation of the same unfolded protein response pathway as previously demonstrated in osteoblasts. Aga2+/- proliferating chondrocytes showed increased FGF and MAPK signaling, findings consistent with accelerated differentiation. There was also increased Sox9 expression throughout the growth plate, which is expected to accelerate early chondrocyte differentiation but reduce late hypertrophic differentiation. These data reveal that mutant type I collagen expression in OI has an impact on the cartilage growth plate. These effects on endochondral ossification indicate that OI is a biologically complex phenotype going beyond its known impacts on bone to negatively affect linear growth.

Hes1 marks peri-condensation mesenchymal cells that generate both chondrocytes and perichondrial cells in early bone development

Bone development starts with condensations of undifferentiated mesenchymal cells that set a framework for future bones within the primordium. In the endochondral pathway, mesenchymal cells inside the condensation differentiate into chondrocytes and perichondrial cells in a SOX9-dependent mechanism. However, the identity of mesenchymal cells outside the condensation and how they participate in developing bones remain undefined. Here we show that mesenchymal cells surrounding the condensation contribute to both cartilage and perichondrium, robustly generating chondrocytes, osteoblasts, and marrow stromal cells in developing bones. Single-cell RNA-seq analysis of Prrx1-cre-marked limb bud mesenchymal cells at E11.5 reveals that Notch effector Hes1 is expressed in a mutually exclusive manner with Sox9 that is expressed in pre-cartilaginous condensations. Analysis of a Notch signaling reporter CBF1:H2B-Venus reveals that peri-condensation mesenchymal cells are active for Notch signaling. Invivo lineage-tracing analysis using Hes1-creER identifies that Hes1+ early mesenchymal cells surrounding the SOX9+ condensation at E10.5 contribute to both cartilage and perichondrium at E13.5, subsequently becoming growth plate chondrocytes, osteoblasts of trabecular and cortical bones, and marrow stromal cells in postnatal bones. In contrast, Hes1+ cells in the perichondrium at E12.5 or E14.5 do not generate chondrocytes within cartilage, contributing to osteoblasts and marrow stromal cells only through the perichondrial route. Therefore, Hes1+ peri-condensation mesenchymal cells give rise to cells of the skeletal lineage through cartilage-dependent and independent pathways, supporting the theory that early mesenchymal cells outside the condensation also play important roles in early bone development.

Hypoxia Differentially Affects Chondrogenic Differentiation of Progenitor Cells from Different Origins.

Ear cartilage malformations are commonly encountered problems in reconstructive surgery, since cartilage has low self-regenerating capacity. Malformations that impose psychological and social burden on one's life are currently treated using ear prosthesis, synthetic implants or autologous flaps from rib cartilage. These approaches are challenging because not only they request high surgical expertise, but also they lack flexibility and induce severe donor-site morbidity. Through the last decade, tissue engineering gained attention where it aims at regenerating human tissues or organs in order to restore normal functions. This technique consists of three main elements, cells, growth factors, and above all, a scaffold that supports cells and guides their behavior. Several studies have investigated different scaffolds prepared from both synthetic or natural materials and their effects on cellular differentiation and behavior. In this study, we investigated a natural scaffold (alginate) as tridimensional hydrogel seeded with progenitors from different origins such as bone marrow, perichondrium and dental pulp. In contact with the scaffold, these cells remained viable and were able to differentiate into chondrocytes when cultured in vitro. Quantitative and qualitative results show the presence of different chondrogenic markers as well as elastic ones for the purpose of ear cartilage, upon different culture conditions. We confirmed that auricular perichondrial cells outperform other cells to produce chondrogenic tissue in normal oxygen levels and we report for the first time the effect of hypoxia on these cells. Our results provide updates for cartilage engineering for future clinical applications.

Good Manufacturing Practice–compliant change of raw material in the manufacturing process of a clinically used advanced therapy medicinal product–a comparability study

The development of medicinal products often continues throughout the different phases of a clinical study and may require challenging changes in raw and starting materials at later stages. Comparability between the product properties pre- and post-change thus needs to be ensured. Here, we describe and validate the regulatory compliant change of a raw material using the example of a nasal chondrocyte tissue-engineered cartilage (N-TEC) product, initially developed for treatment of confined knee cartilage lesions. Scaling up the size of N-TEC as required for the treatment of larger osteoarthritis defects required the substitution of autologous serum with a clinical-grade human platelet lysate (hPL) to achieve greater cell numbers necessary for the manufacturing of larger size grafts. A risk-based approach was performed to fulfill regulatory requirements and demonstrate comparability of the products manufactured with the standard process (autologous serum) already applied in clinical indications and the modified process (hPL). Critical attributes with regard to quality, purity, efficacy, safety and stability of the product as well as associated test methods and acceptance criteria were defined. Results showed that hPL added during the expansion phase of nasal chondrocytes enhances proliferation rate, population doublings and cell numbers at passage 2 without promoting the overgrowth of potentially contaminant perichondrial cells. N-TEC generated with the modified versus standard process contained similar content of DNA and cartilaginous matrix proteins with even greater expression levels of chondrogenic genes. The increased risk for tumorigenicity potentially associated with the use of hPL was assessed through karyotyping of chondrocytes at passage 4, revealing no chromosomal changes. Moreover, the shelf-life of N-TEC established for the standard process could be confirmed with the modified process. In conclusion, we demonstrated the introduction of hPL in the manufacturing process of a tissue engineered product, already used in a late-stage clinical trial. Based on this study, the national competent authorities in Switzerland and Germany accepted the modified process which is now applied for ongoing clinical tests of N-TEC. The described activities can thus be taken as a paradigm for successful and regulatory compliant demonstration of comparability in advanced therapy medicinal products manufacturing.

The fate of early perichondrial cells in developing bones

In endochondral bone development, bone-forming osteoblasts and bone marrow stromal cells have dual origins in the fetal cartilage and its surrounding perichondrium. However, how early perichondrial cells distinctively contribute to developing bones remain unidentified. Here we show using in vivo cell-lineage analyses that Dlx5+ fetal perichondrial cells marked by Dlx5-creER do not generate cartilage but sustainably contribute to cortical bone and marrow stromal compartments in a manner complementary to fetal chondrocyte derivatives under the regulation of Hedgehog signaling. Postnatally, Dlx5+ fetal perichondrial cell derivatives preferentially populate the diaphyseal marrow stroma with a dormant adipocyte-biased state and are refractory to parathyroid hormone-induced bone anabolism. Therefore, early perichondrial cells of the fetal cartilage are destined to become an adipogenic subset of stromal cells in postnatal diaphyseal bone marrow, supporting the theory that the adult bone marrow stromal compartments are developmentally prescribed within the two distinct cells-of-origins of the fetal bone anlage.

Creb5 coordinates synovial joint formation with the genesis of articular cartilage

While prior work has established that articular cartilage arises from Prg4-expressing perichondrial cells, it is not clear how this process is specifically restricted to the perichondrium of synovial joints. We document that the transcription factor Creb5 is necessary to initiate the expression of signaling molecules that both direct the formation of synovial joints and guide perichondrial tissue to form articular cartilage instead of bone. Creb5 promotes the generation of articular chondrocytes from perichondrial precursors in part by inducing expression of signaling molecules that block a Wnt5a autoregulatory loop in the perichondrium. Postnatal deletion of Creb5 in the articular cartilage leads to loss of both flat superficial zone articular chondrocytes coupled with a loss of both Prg4 and Wif1 expression in the articular cartilage; and a non-cell autonomous up-regulation of Ctgf. Our findings indicate that Creb5 promotes joint formation and the subsequent development of articular chondrocytes by driving the expression of signaling molecules that both specify the joint interzone and simultaneously inhibit a Wnt5a positive-feedback loop in the perichondrium.

GATA-3 in Atlantic salmon (Salmo salar): Tissue distribution and its regulation of IL-4/13a promoter.

GATA3 is a transcription factor that plays an important role in T cell lineage differentiation and T-helper 2 (Th2) type immune responses. In this study, we developed two rat antibodies against Atlantic salmon GATA-3 (anti-rSsGATA-3a and anti-rSsGATA-3b, respectively). The western blotting and immunofluorescence results showed that anti-rSsGATA-3b antibodies recognized endogenous SsGATA-3 proteins, while the anti-rSsGATA-3a antibodies did not bind SsGATA-3. Immunohistochemical analysis revealed that SsGATA-3 positive cells were detected in all tissues tested, with relatively high number of immune reactive cells in the gills and spleen. Furthermore, the immunohistochemical study revealed that SsGATA-3 was expressed in pillar cells, epithelial cells, chondrocytes, perichondrium cells, and some undifferentiated basal cells. In addition, we determined 577 bp of the upstream promoter sequence of SsIL-4/13a and found four motifs that matched SsGATA-3 binding sites. The promoter regions of SsIL-4/13a were assessed by transfecting four deletion reporter constructs and SsGATA-3 overexpression plasmids. The result showed that SsGATA-3 enhanced the activity of SsIL-4/13a promoters within the region ranging from -317 to -302 bp upstream of the transcriptional start site. Antibodies against Th2 markers such as GATA-3 are valuable in addressing the diversity of T cell responses in fish.

Innovative mouse models for the tumor suppressor activity of Protocadherin-10 isoforms

BackgroundNonclustered mouse protocadherin genes (Pcdh) encode proteins with a typical single ectodomain and a cytoplasmic domain with conserved motifs completely different from those of classic cadherins. Alternative splice isoforms differ in the size of these cytoplasmic domains. In view of the compelling evidence for gene silencing of protocadherins in human tumors, we started investigations on Pcdh functions in mouse cancer models.MethodsFor Pcdh10, we generated two mouse lines: one with floxed exon 1, leading to complete Pcdh10 ablation upon Cre action, and one with floxed exons 2 and 3, leading to ablation of only the long isoforms of Pcdh10. In a mouse medulloblastoma model, we used GFAP-Cre action to locally ablate Pcdh10 in combination with Trp53 and Rb1 ablation. From auricular tumors, that also arose, we obtained tumor-derived cell lines, which were analyzed for malignancy in vitro and in vivo. By lentiviral transduction, we re-expressed Pcdh10 cDNAs. RNA-Seq analyses were performed on these cell families.ResultsSurprisingly, not only medulloblastomas were generated in our model but also tumors of tagged auricles (pinnae). For both tumor types, ablation of either all or only long isoforms of Pcdh10 aggravated the disease. We argued that the perichondrial stem cell compartment is at the origin of the pinnal tumors. Immunohistochemical analysis of these tumors revealed different subtypes. We obtained several pinnal-tumor derived (PTD) cell lines and analyzed these for anchorage-independent growth, invasion into collagen matrices, tumorigenicity in athymic mice. Re-expression of either the short or a long isoform of Pcdh10 in two PTD lines counteracted malignancy in all assays. RNA-Seq analyses of these two PTD lines and their respective Pcdh10-rescued cell lines allowed to identify many interesting differentially expressed genes, which were largely different in the two cell families.ConclusionsA new mouse model was generated allowing for the first time to examine the remarkable tumor suppression activity of protocadherin-10 in vivo. Despite lacking several conserved motifs, the short isoform of Pcdh10 was fully active as tumor suppressor. Our model contributes to scrutinizing the complex molecular mechanisms of tumor initiation and progression upon PCDH10 silencing in many human cancers.

Perichondrial progenitor cells promote proliferation and chondrogenesis of mature chondrocytes.

Autologous chondrocytes (C cells) are effective sources of cell therapy for engineering cartilage tissue to repair chondral defects, such as degenerative arthritis. The expansion of cells with C cell characteristics has become a major challenge due to inadequate donor sites and poor proliferation of mature C cells. The perichondrial progenitor cells (P cells) from the cambium layer of the perichondrium possessed significantly higher mesenchymal stem cell markers than C cells. In the transwell co-culture system, P cells increased the passaging capacity of C cells from P6 to P9, and the cell number increased 128 times. This system increased the percentage of Alcian blue-positive C cells from 40% in P6 to 62% in P9, contributing about 198 times more Alcian blue-positive C cells than the control group. C cells co-cultured with P cells also exhibited higher proliferation than C cells cultured with P cell-conditioned medium. Similar results were obtained in nude mice that were subcutaneously implanted with C cells, P cells or a mixture of the two cell types, in which the presence of both cells enhanced neocartilage formation in vivo. In aggregate, P cells enhanced the proliferation of C cells in a dose–dependent manner and prolonged the longevity of mature C cells for clinical applications.

In vitro elastic cartilage reconstruction using human auricular perichondrial chondroprogenitor cell–derived micro 3D spheroids

Morphologically stable scaffold-free elastic cartilage tissue is crucial for treating external ear abnormalities. However, establishing adequate mechanical strength is challenging, owing to the difficulty of achieving chondrogenic differentiation in vitro; thus, cartilage reconstruction is a complex task. Auricular perichondrial chondroprogenitor cells exhibit high proliferation potential and can be obtained with minimal invasion. Therefore, these cells are an ideal resource for elastic cartilage reconstruction. In this study, we aimed to develop a novel in vitro scaffold-free method for elastic cartilage reconstruction, using human auricular perichondrial chondroprogenitor cells. Inducing chondrogenesis by using microscopic spheroids similar to auricular hillocks significantly increased the chondrogenic potential. The size and elasticity of the tissue were maintained after craniofacial transplantation in immunodeficient mice, suggesting that the reconstructed tissue was morphologically stable. Our novel tissue reconstruction method may facilitate the development of future treatments for external ear abnormalities.

Rat perichondrium transplanted to articular cartilage defects forms articular-like, hyaline cartilage.

Perichondrium autotransplants have been used to reconstruct articular surfaces destroyed by infection or trauma. However, the role of the transplanted perichondrium in the healing of resurfaced joints has not been investigated. Perichondrial and periosteal tissues were harvested from rats hemizygous for a ubiquitously expressed enhanced green fluorescent protein (EGFP) transgene and transplanted into full-thickness articular cartilage defects at the trochlear groove of distal femur in wild-type littermates. As an additional control, cartilage defects were left without a transplant (no transplant control). Distal femurs were collected 3, 14, 56, 112days after surgery. Tracing of transplanted cells showed that both perichondrium and periosteum transplant-derived cells made up the large majority of the cells in the regenerated joint surfaces. Perichondrium transplants contained SOX9 positive cells and with time differentiated into a hyaline cartilage that expanded and filled out the defects with Col2a1-positive and Col1a1-negative chondrocytes and a matrix rich in proteoglycans. At later timepoints the cartilaginous perichondrium transplants were actively remodeled into bone at the transplant-bone interface and at post-surgery day 112 EGFP-positive perichondrium cells at the articular surface were positive for Prg4. Periosteum transplants initially lacked SOX9 expression and despite a transient increase in SOX9 expression and chondrogenic differentiation, remained Col1a1 positive, and were continuously thinning as periosteum-derived cells were incorporated into the subchondral compartment. Perichondrium and periosteum transplanted to articular cartilage defects did not just stimulate regeneration but were themselves transformed into cartilaginous articular surfaces. Perichondrium transplants developed into an articular-like, hyaline cartilage, whereas periosteum transplants appeared to produce a less resilient fibro-cartilage.

Rat perichondrium transplanted to articular cartilage defects forms articular-like, hyaline cartilage

Abstract Objective Perichondrium autotransplants have been used to reconstruct articular surfaces destroyed by infection or trauma. However, the role of the transplanted perichondrium in the healing of resurfaced joints has not been investigated. Design Perichondrial and periosteal tissues were harvested from rats hemizygous for a ubiquitously expressed enhanced green fluorescent protein (EGFP) transgene and transplanted into full-thickness articular cartilage defects at the trochlear groove of distal femur in wild-type littermates. As an additional control, cartilage defects were left without a transplant (no transplant control). Distal femurs were collected 3, 14, 56, 112 days after surgery. Results Tracing of transplanted cells showed that both perichondrium and periosteum transplant-derived cells made up the large majority of the cells in the regenerated joint surfaces. Perichondrium transplants contained SOX9 positive cells and with time differentiated into a hyaline cartilage that expanded and filled out the defects with Col2a1-positive and Col1a1-negative chondrocytes and a matrix rich in proteoglycans. At later timepoints the cartilaginous perichondrium transplants were actively remodeled into bone at the transplant-bone interface and at post-surgery day 112 EGFP-positive perichondrium cells at the articular surface were positive for Prg4. Periosteum transplants initially lacked SOX9 expression and despite a transient increase in SOX9 expression and chondrogenic differentiation, remained Col1a1 positive, and were continuously thinning as periosteum-derived cells were incorporated into the subchondral compartment. Conclusions Perichondrium and periosteum transplanted to articular cartilage defects did not just stimulate regeneration but were themselves transformed into cartilaginous articular surfaces. Perichondrium transplants developed into an articular-like, hyaline cartilage, whereas periosteum transplants appeared to produce a less resilient fibro-cartilage.

MMP-9 Expression in Normal Rabbit Chondrocytes

Chondrocytes regulate anabolic and catabolic processes to maintain the extracellular matrix components. Catabolic activities depend on the proteolytic action of the matrix -degrading enzymes including ADAMTS (A disintegrin and metalloproteinases) and MMP (Matrix Metalloproteinase). The current study explored the distribution of MMP-9 in normal articular cartilages of the embryos rabbit. Articular cartilage has grown by appositional growth that the perichondrial stem cells differentiate into chondrocytes. MMP-9 positive perichondrial stem cells or chondroblasts and early chondrocytes. Mature chondrocytes exhibited weak immunoaffinity for MMP-9. In conclusion, MMP-9 was essential during chondrocytes growth. The current study alludes to the potential role of MMP-9 during the growth of the articular cartilage.

Tendon Cell Regeneration Is Mediated by Attachment Site-Resident Progenitors and BMP Signaling

The musculoskeletal system is a striking example of how cell identity and position is coordinated across multiple tissues to ensure function. However, it is unclear upon tissue loss, such as complete loss of cells of a central musculoskeletal connecting tendon, whether neighboring tissues harbor progenitors capable of mediating regeneration. Here, using a zebrafish model, we genetically ablate all embryonic tendon cells and find complete regeneration of tendon structure and pattern. We identify two regenerative progenitor populations, sox10+ perichondrial cells surrounding cartilage and nkx2.5+ cells surrounding muscle. Surprisingly, laser ablation of sox10+ cells, but not nkx2.5+ cells, increases tendon progenitor number in the perichondrium, suggesting a mechanism to regulate attachment location. We find BMP signaling is active in regenerating progenitor cells and is necessary and sufficient for generating new scxa+ cells. Our work shows that muscle and cartilage connective tissues harbor progenitor cells capable of fully regenerating tendons, and this process is regulated by BMP signaling.

Biomarker Signatures of Quality for Engineering Nasal Chondrocyte-Derived Cartilage.

The definition of quality controls for cell therapy and engineered product manufacturing processes is critical for safe, effective, and standardized clinical implementation. Using the example context of cartilage grafts engineered from autologous nasal chondrocytes, currently used for articular cartilage repair in a phase II clinical trial, we outlined how gene expression patterns and generalized linear models can be introduced to define molecular signatures of identity, purity, and potency. We first verified that cells from the biopsied nasal cartilage can be contaminated by cells from a neighboring tissue, namely perichondrial cells, and discovered that they cannot deposit cartilaginous matrix. Differential analysis of gene expression enabled the definition of identity markers for the two cell populations, which were predictive of purity in mixed cultures. Specific patterns of expression of the same genes were significantly correlated with cell potency, defined as the capacity to generate tissues with histological and biochemical features of hyaline cartilage. The outlined approach can now be considered for implementation in a good manufacturing practice setting, and offers a paradigm for other regenerative cellular therapies.

Growth plate skeletal stem cells and their transition from cartilage to bone.

The growth plate is an essential component of endochondral bone development. Not surprisingly, the growth plate and its surrounding structure, the perichondrium, contain a wealth of skeletal stem cells (SSCs) and progenitor cells that robustly contribute to bone development. Recent in vivo lineage-tracing studies using mouse genetic models provide substantial insight into the diversity and versatility of these skeletal stem and progenitor cell populations, particularly shedding light on the importance of the transition from cartilage to bone. Chondrocytes and perichondrial cells are inseparable twins that develop from condensing undifferentiated mesenchymal cells during the fetal stage; although morphologically and functionally distinct, these cells ultimately serve for the same goal, that is, to make bone bigger and stronger. Even in the postnatal stage, a small subset of growth plate chondrocytes can transform into osteoblasts and marrow stromal cells; this is in part fueled by a unique type of SSCs maintained in the resting zone of the growth plate, which continue to self-renew for the long term. Here, we discuss diverse skeletal stem and progenitor cell populations in the growth plate and the perichondrium and their transition from cartilage to bone.

Engineering of scaffold-free tri-layered auricular tissues for external ear reconstruction.

Current strategies for external ear reconstruction can lead to donor site morbidity and/or surgical complications. Tissue-engineered auricular tissues may provide readily available reconstructive materials that resemble native auricular tissue, which is composed of a cartilaginous region sandwiched between two perichondrial layers. We previously developed scaffold-free bi-layered auricular tissues, consisting of a perichondrial layer and a cartilaginous layer, by cultivating chondrocytes and perichondrial cells in a continuous flow bioreactor. Here, we aimed to improve construct properties and develop strategies to engineer tri-layered auricular constructs that better mimic native auricular tissue. Experimental study. Different concentrations of insulin-like growth factor (IGF)-1 and insulin were supplemented during bioreactor culture to determine conditions for engineering bi-layered constructs. We also investigated two methods of engineering tri-layered constructs. Method 1 used Ficoll separation to isolate perichondrial cells, followed by the seeding of isolated perichondrial cells onto the opposing side of the bi-layered constructs. Method 2 involved the growth of the bi-layered constructs in osteogenic culture medium. The combination of 10 nM IGF-1 and 100 nM insulin led to increased collagen content in the engineered bi-layered constructs. For developing tri-layered constructs, method 2 yielded thicker constructs with better mechanical and biochemical properties compared to method 1. In addition, the presence of the perichondrial layers protected the engineered constructs from tissue calcification. Auricular tissues with a biomimetic microstructure can be created by growing chondrocytes and perichondrial cells in a continuous flow bioreactor, followed by cultivation in osteogenic medium. NA Laryngoscope, 129:E272-E283, 2019.

Evidence That Rat Chondrocytes Can Differentiate Into Perichondrial Cells.

ABSTRACTDuring early bone formation, mesenchymal cells condense and then differentiate into collagen type II‐expressing chondrocytes that make up the cartilaginous bone anlagen. This anlage then becomes enclosed by the perichondrium. The mechanisms by which the perichondrium forms are not known. The purpose of this study was to determine whether epiphyseal chondrocytes can differentiate into perichondrial cells. Novel perichondrium markers were identified by expression microarray of microdissected rat perichondrium and growth plate cartilage. A dissection method that allowed for removal of contaminating perichondrium was developed and the absence was confirmed by histological examination and by expression of perichondrium markers. Perichondrium formation surrounding chondrocyte pellets was studied using histology, real‐time PCR, and in situ hybridization for chondrocyte and perichondrium markers. Cultured chondrocyte pellets developed an exterior perichondrium‐like layer. This surrounding tissue did not express chondrocyte markers, collagen‐type II and type X, as assessed by in situ hybridization. Instead, perichondrium markers, periostin, Dickkopf 3 (Dkk3), roundabout 2, cadherin 2, L‐galectin 1 (Lgals1), and thrombospondin 2 (Thbs2) were upregulated following formation of the perichondrium‐like layer as assessed by real‐time PCR. Interestingly, markers specific for the cambium layer, Dkk3, Thbs2, and Lgals1, but not for the fibrous layer, collagen‐type XIV and decorin, were upregulated. The findings suggest that epiphyseal chondrocytes of postnatal animals retain the potential to differentiate into perichondrial cells, supporting the hypothesis that the perichondrium originates from collagen type II‐expressing chondrocytes at the periphery of the cartilaginous bone template. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Msx2 Marks Spatially Restricted Populations of Mesenchymal Precursors

Craniofacial development requires a set of patterning codes that define the identities of postmigratory mesenchymal cells in a region-specific manner, in which locally expressed morphogens, including fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs), provide instructive cues. Msx2, a bona fide target of BMP signaling, is a transcription factor regulating Runx2 and osterix (Osx), whose mutations are associated with cranial deformities in humans. Here we show that Msx2 defines osteo-chondro precursor cells in specific regions of the craniofacial mesenchyme at the postmigratory stage, particularly in the mandibular process and the posterior cranial vault. Analysis of Msx2-creER mice revealed that early mesenchymal cells in proximity to the BMP4-expressing mesenchyme were marked upon tamoxifen injection, and their descendants contributed to diverse types of mesenchymal cells in the later stage, such as chondrocytes and perichondrial cells of the transient cartilage, as well as osteoblasts and suture mesenchymal cells. By contrast, Osx-creER marked osteoblast precursors at the later stage, and their descendants continued to become osteoblasts well into the postnatal stage. Therefore, Msx2 marks spatially restricted populations of mesenchymal precursor cells with diverse differentiation potential, suggesting that extrinsic molecular cues can dictate the nature of postmigratory mesenchymal cells in craniofacial development.