Identification of Mechanosensitive Genes during Embryonic Bone Formation

Although it is known that mechanical forces are needed for normal bone development, the current understanding of how biophysical stimuli are interpreted by and integrated with genetic regulatory mechanisms is limited. Mechanical forces are thought to be mediated in cells by “mechanosensitive” genes, but it is a challenge to demonstrate that the genetic regulation of the biological system is dependant on particular mechanical forces in vivo. We propose a new means of selecting candidate mechanosensitive genes by comparing in vivo gene expression patterns with patterns of biophysical stimuli, computed using finite element analysis. In this study, finite element analyses of the avian embryonic limb were performed using anatomically realistic rudiment and muscle morphologies, and patterns of biophysical stimuli were compared with the expression patterns of four candidate mechanosensitive genes integral to bone development. The expression patterns of two genes, Collagen X (ColX) and Indian hedgehog (Ihh), were shown to colocalise with biophysical stimuli induced by embryonic muscle contractions, identifying them as potentially being involved in the mechanoregulation of bone formation. An altered mechanical environment was induced in the embryonic chick, where a neuromuscular blocking agent was administered in ovo to modify skeletal muscle contractions. Finite element analyses predicted dramatic changes in levels and patterns of biophysical stimuli, and a number of immobilised specimens exhibited differences in ColX and Ihh expression. The results obtained indicate that computationally derived patterns of biophysical stimuli can be used to inform a directed search for genes that may play a mechanoregulatory role in particular in vivo events or processes. Furthermore, the experimental data demonstrate that ColX and Ihh are involved in mechanoregulatory pathways and may be key mediators in translating information from the mechanical environment to the molecular regulation of bone formation in the embryo.     

Dynamic patterns of mechanical stimulation co-localise with growth and cell proliferation during morphogenesis in the avian embryonic knee joint

Muscle contractions begin in early embryonic life, generating forces that regulate the correct formation of the skeleton. In this paper we test the hypothesis that the biophysical stimulation generated by muscle forces may be a causative factor for the changes in shape of the knee joint as it grows. We do this by predicting the spatial and temporal patterns of biophysical stimuli, where cell proliferation and rudiment shape changes occur within the emerging tissues of the joint over time. We used optical projection tomography (OPT) to create anatomically accurate finite element models of the embryonic knee at three time points (stages) of development. OPT was also used to locate muscle attachment sites and AFM was used to determine material properties. An association was found between the emergence of joint shape, cell proliferation and the pattern of biophysical stimuli generated by embryonic muscle contractions. Elevated rates of growth and cell proliferation in the medial condyle were found to co-localise with elevated patterns of biophysical stimuli including maximum principal stresses and fluid flow, throughout the time period studied, indicating that cartilage growth and chondrocyte proliferation in the epiphysis is potentially related to local patterns of biophysical stimuli. The development of the patella and articular cartilages, which is known to be affected by in ovo immobilisation, could be contributed to by specific patterns of fluid flow, pore pressure and stress in the joint interzone. This suggests that both cartilage growth and tissue differentiation in the embryonic joint is regulated by specific patterns of biophysical stimuli and that these stimuli are needed for the correct development of the joint.     

Isolation and characterization of osteogenic cells derived from first bone of the embryonic tibia

Osteogenesis in the embryonic long bone rudiment occurs initially within an outer periosteal membrane and subsequently inside the cartilaginous core as a consequence of the endochondral ossification process. In order to investigate the development of these two different mechanisms of bone formation, embryonic chick tibial cell isolates were prepared from sites of first periosteal bone formation and from the immediately underlying hypertrophic cartilaginous core region. Mid-diaphyseal periosteal collars and the corresponding cartilage core were microdissected free from Hamburger-Hamilton stage 35 (Day 9) chick tibias and separately digested with a trypsin-collagenase enzyme mixture. The released cell populations were cultivated in vitro and characterized by morphological analysis, histochemical localization of alkaline phosphatase, alizarin red S staining for mineral deposition, growth rate [( 3H]thymidine uptake), and proteoglycan content. Results of these studies showed that periosteal collar cell cultures form nodule-like structures that stain positive with alkaline phosphatase and alizarin red S. Light and electron microscopic observation revealed cell and matrix morphologies similar to that of intact periosteum. The nodules were composed of plump cell types embedded within a mineralized matrix surrounded by a fibroblastic cell layer. Core cartilage cell cultures displayed typical characteristics of the hypertrophic state in their visual appearance and proteoglycan composition. The formation of osseous-like structures in periosteal collar cell cultures but not in core chondrocyte cell cultures demonstrates the relatively autonomous nature of intramembranous ossification while emphasizing the dependence of the endochondral ossification process upon an intact vascularized environment present in the developing tibia.     

Development and regeneration of the neonatal digit tip in mice

The digit tips of children and rodents are known to regenerate following amputation. The skeletal structure that regenerates is the distal region of the terminal phalangeal bone that is associated with the nail organ. The terminal phalanx forms late in gestation by endochondral ossification and continues to elongate until sexual maturity (8 weeks of age). Postnatal elongation at its distal end occurs by appositional ossification, i.e. direct ossification on the surface of the terminal phalanx, whereas proximal elongation results from an endochondral growth plate. Amputation through the middle of the terminal phalanx regenerates whereas regenerative failure is observed following amputation to remove the distal 2/3 of the bone. Regeneration is characterized by the formation of a blastema of proliferating cells that appear undifferentiated and express Bmp4. Using chondrogenic and osteogenic markers we show that redifferentiation does not occur by endochondral ossification but by the direct ossification of blastema cells that form the rudiment of the digit tip. Once formed the rudiment elongates by appositional ossification in parallel with unamputated control digits. Regenerated digits are consistently shorter than unamputated control digits. Finally, we present a case study of a child who suffered an amputation injury at a proximal level of the terminal phalanx, but failed to regenerate despite conservative treatment and the presence of the nail organ. These clinical and experimental findings expand on previously published observations and initiate a molecular assessment of a mammalian regeneration model.     

Mechanobiological modeling of endochondral ossification: an experimental and computational analysis

Long bone formation starts early during embryonic development through a process known as endochondral ossification. This is a highly regulated mechanism that involves several mechanical and biochemical factors. Because long bone development is an extremely complex process, it is unclear how biochemical regulation is affected when dynamic loads are applied, and also how the combination of mechanical and biochemical factors affect the shape acquired by the bone during early development. In this study, we develop a mechanobiological model combining: (1) a reaction–diffusion system to describe the biochemical process and (2) a poroelastic model to determine the stresses and fluid flow due to loading. We simulate endochondral ossification and the change in long bone shapes during embryonic stages. The mathematical model is based on a multiscale framework, which consisted in computing the evolution of the negative feedback loop between Ihh/PTHrP and the diffusion of VEGF molecule (on the order of days) and dynamic loading (on the order of seconds). We compare our morphological predictions with the femurs of embryonic mice. The results obtained from the model demonstrate that pattern formation of Ihh, PTHrP and VEGF predict the development of the main structures within long bones such as the primary ossification center, the bone collar, the growth fronts and the cartilaginous epiphysis. Additionally, our results suggest high load pressures and frequencies alter biochemical diffusion and cartilage formation. Our model incorporates the biochemical and mechanical stimuli and their interaction that influence endochondral ossification during embryonic growth. The mechanobiochemical framework allows us to probe the effects of molecular events and mechanical loading on development of bone.     

Mechanical influences on morphogenesis of the knee joint revealed through morphological, molecular and computational analysis of immobilised embryos

Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint.     

Developmental control of chondrogenesis and osteogenesis

During vertebrate embryogenesis, bones of the vertebral column, pelvis, and upper and lower limbs, are formed on an initial cartilaginous model. This process, called endochondral ossification, is characterized by a precise series of events such as aggregation and differentiation of mesenchymal cells, and proliferation, hypertrophy and death of chondrocytes. Bone formation initiates in the collar surrounding the hypertrophic cartilage core that is eventually invaded by blood vessels and replaced by bone tissue and bone marrow. Over the last years we have extensively investigated cellular and molecular events leading to cartilage and bone formation. This has been partially accomplished by using a cell culture model developed in our laboratory. In several cases observations have been confirmed or directly made in the developing embryonic bone of normal and genetically modified chick and mouse embryos. In this article we will review our work in this field.     

Influence of electromagnetic fields on endochondral bone formation

Endochondral ossification is a basic physiological process in limb development and is central to bone repair and linear growth. Factors which regulate endochondral ossification include several biophysical and biochemical agents and are of interest from clinical and biological perspectives. One of these agents, electric stimulation, has been shown to result in enhanced synthesis of extracellular matrix, calcification, and bone formation in a number of experimental systems and is the subject of this review. The effects of electric stimulation have been studied in embryonic limb rudiments, growth plates, and experimental endochondral ossification induced with decalcified bone matrix and, in all these models, endochondral ossification has been enhanced. It is not known definitively whether electric fields stimulate cell differentiation or modulate an increased number of molecules synthesized by committed cell population and this is a fertile area of current study.     

An experimental analysis of factors affecting the localization of embryonic bone marrow

Summary The present investigations have been concerned with factors which determine and influence the localization and development of hemopoietic bone marrow in the embryo mouse and the adult. These studies, which have employed organ cultures and the transplantation of mouse embryo femur and tail rudiments, indicate that the surrounding mesenchyme is required for the normal development of the cartilage rudiment and its ossification, and for the formation and colonization of the marrow cavity. It was suggested that hemopoiesis results from the colonization of the “prepared” marrow cavity by stem cells arising from sources external to the rudiment. The addition of erythropoietin and L-thyroxine produced distinct erythropoietic differentiation in the normally myelocytic embryonic marrow cavity. The significance of the microenvironment present in developing bone rudiments and the initiation of hemopoiesis in stem cells was discussed. A hypothesis was developed to explain marrow localization in adults based on the colonization of bone rudiments which are developing their marrow sites at a time when the blood contains large numbers of colony-forming units.     

Biophysical stimuli induced by passive movements compensate for lack of skeletal muscle during embryonic skeletogenesis

In genetically modified mice with abnormal skeletal muscle development, bones and joints are differentially affected by the lack of skeletal muscle. We hypothesise that unequal levels of biophysical stimuli in the developing humerus and femur can explain the differential effects on these rudiments when muscle is absent. We find that the expression patterns of four mechanosensitive genes important for endochondral ossification are differentially affected in muscleless limb mutants, with more extreme changes in the expression in the humerus than in the femur. Using finite element analysis, we show that the biophysical stimuli induced by muscle forces are similar in the humerus and femur, implying that the removal of muscle contractile forces should, in theory, affect the rudiments equally. However, simulations in which a displacement was applied to the end of the limb, such as could be caused in muscleless mice by movements of the mother or normal littermates, predicted higher biophysical stimuli in the femur than in the humerus. Stimuli induced by limb movement were much higher than those induced by the direct application of muscle forces, and we propose that movements of limbs caused by muscle contractions, rather than the direct application of muscle forces, provide the main mechanical stimuli for normal skeletal development. In muscleless mice, passive movement induces unequal biophysical stimuli in the humerus and femur, providing an explanation for the differential effects seen in these mice. The significance of these results is that forces originating external to the embryo may contribute to the initiation and progression of skeletal development when muscle development is abnormal.     

L-Maf,a downstream target of Pax6, is essential for chick lens development

Vascular endothelial growth factor (VEGF)-mediated angiogenesis is an important part of bone formation. To clarify the role of VEGF isoforms in endochondral bone formation, we examined long bone development in mice expressing exclusively the VEGF120 isoform (VEGF120/120 mice). Neonatal VEGF120/120 long bones showed a completely disturbed vascular pattern, concomitant with a 35% decrease in trabecular bone volume, reduced bone growth and a 34% enlargement of the hypertrophic chondrocyte zone of the growth plate. Surprisingly, embryonic hindlimbs at a stage preceding capillary invasion exhibited a delay in bone collar formation and hypertrophic cartilage calcification. Expression levels of marker genes of osteoblast and hypertrophic chondrocyte differentiation were significantly decreased in VEGF120/120 bones. Furthermore, inhibition of all VEGF isoforms in cultures of embryonic cartilaginous metatarsals, through the administration of a soluble receptor chimeric protein (mFlt-1/Fc), retarded the onset and progression of ossification, suggesting that osteoblast and/or hypertrophic chondrocyte development were impaired. The initial invasion by osteoclasts and endothelial cells into VEGF120/120 bones was retarded, associated with decreased expression of matrix metalloproteinase-9. Our findings indicate that expression of VEGF164 and/or VEGF188 is important for normal endochondral bone development, not only to mediate bone vascularization but also to allow normal differentiation of hypertrophic chondrocytes, osteoblasts, endothelial cells and osteoclasts.     

Wound healing and blastema formation in regenerating digit tips of adult mice

Amputation of the distal region of the terminal phalanx of mice causes an initial wound healing response followed by blastema formation and the regeneration of the digit tip. Thus far, most regeneration studies have focused in embryonic or neonatal models and few studies have examined adult digit regeneration. Here we report on studies that include morphological, immunohistological, and volumetric analyses of adult digit regeneration stages. The regenerated digit is grossly similar to the original, but is not a perfect replacement. Re-differentiation of the digit tip occurs by intramembranous ossification forming a trabecular bone network that replaces the amputated cortical bone. The digit blastema is comprised of proliferating cells that express vimentin, a general mesenchymal marker, and by comparison to mature tissues, contains fewer endothelial cells indicative of reduced vascularity. The majority of blastemal cells expressing the stem cell marker SCA-1, also co-express the endothelial marker CD31, suggesting the presence of endothelial progenitor cells. Epidermal closure during wound healing is very slow and is characterized by a failure of the wound epidermis to close across amputated bone. Instead, the wound healing phase is associated with an osteoclast response that degrades the stump bone allowing the wound epidermis to undercut the distal bone resulting in a novel re-amputation response. Thus, the regeneration process initiates from a level that is proximal to the original plane of amputation.     

Bone growth aud development of secondary ossification centers of extremities in the cynomolgus monkey (Macaca fascicularis).

The development of so-called long bones in the extremity has been studied roentgenographically in forty-seven males and fifty-one females cynomolgus monkeys bred and reared at the National Institute of Health. The age of the females ranged from five months to eight years and nine months, and that of the males was from four months to seven years. In addition, the fetuses of six to twenty weeks of gestation age were examined for the time of appearance of ossification centers. As the biological parameters concerning body growth, the body weight and the bone length were measured and the secondary ossification centers were scrutinized and assessed the maturity process on the basis of the criteria that divided the state into eleven stages. Also the allometric analyses of body weight against bone length was conducted. Most of the secondary ossification centers except the proximal fibulal epiphysis appeared during the period from the prenatal stage (15-20 weeks of gestationage) to the postnatal one (several months of age). From four to five months of age, many ossification centers had developed to some extent. But, the appearance of proximal fibulal epiphysis was delayed and often lacking until 10 months of age in female and one year and three months of age in male. The earliest epiphyseal fusion was observed at the distal humeral epiphysis in both sexes. The latest epiphyseal fusion was observed at the distal ulnal epiphysis in both sexes and at the distal ulnal and radial epiphyses in female. From this study, the time of fusion was at five and three guarters years of age in females and at six and a half years of age in males. As a result, it is suggested that the estimation of animal's age might be put to practical use by introducing the assessing method that the score was given from the observation of the secondary ossification center.     

Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation

Pbx1 and a subset of homeodomain proteins collaboratively bind DNA as higher-order molecular complexes with unknown consequences for mammalian development. Pbx1 contributions were investigated through characterization of Pbx1-deficient mice. Pbx1 mutants died at embryonic day 15/16 with severe hypoplasia or aplasia of multiple organs and widespread patterning defects of the axial and appendicular skeleton. An obligatory role for Pbx1 in limb axis patterning was apparent from malformations of proximal skeletal elements, but distal structures were unaffected. In addition to multiple rib and vertebral malformations, neural crest cell-derived skeletal structures of the second branchial arch were morphologically transformed into elements reminiscent of first arch-derived cartilages. Although the skeletal malformations did not phenocopy single or compound Hox gene defects, they were restricted to domains specified by Hox proteins bearing Pbx dimerization motifs and unaccompanied by alterations in Hox gene expression. In affected domains of limbs and ribs, chondrocyte proliferation was markedly diminished and there was a notable increase of hypertrophic chondrocytes, accompanied by premature ossification of bone. The pattern of expression of genes known to regulate chondrocyte differentiation was not perturbed in Pbx1-deficient cartilage at early days of embryonic skeletogenesis, however precocious expression of Col1a1, a marker of bone formation, was found. These studies demonstrate a role for Pbx1 in multiple developmental programs and reveal a novel function in co-ordinating the extent and/or timing of proliferation with terminal differentiation. This impacts on the rate of endochondral ossification and bone formation and suggests a mechanistic basis for most of the observed skeletal malformations.     

A semi-empirical cell dynamics model for bone turnover under external stimulus

The normal periodic turnover of bone is referred to as remodeling. In remodeling, old or damaged bone is removed during a 'resorption' phase and new bone is formed in its place during a 'formation' phase in a sequence of events known as coupling. Resorption is preceded by an 'activation' phase in which the signal to remodel is initiated and transmitted. Remodeling is known to involve the interaction of external stimuli, bone cells, calcium and phosphate ions, and several proteins, hormones, molecules, and factors. In this study, a semi-empirical cell dynamics model for bone remodeling under external stimulus that accounts for the interaction between bone mass, bone fluid calcium, bone calcium, and all three major bone cell types, is presented. The model is formulated to mimic biological coupling by solving separately and sequentially systems of ODEs for the activation, resorption, and formation phases of bone remodeling. The charateristic time for resorption (20 days) and the amount of resorption (~0.5%) are fixed for all simulations, but the formation time at turnover is an output of the model. The model was used to investigate the effects of different types of strain stimuli on bone turnover under bone fluid calcium balance and imbalance conditions. For bone fluid calcium balance, the model predicts complete turnover after 130 days of formation under constant 1000 microstrain stimulus; after 47 days of formation under constant 2000 microstrain stimulus; after 173 days of formation under strain-free conditions, and after 80 days of formation under monotonic increasing strain stimulus from 1000 to 2000 microstrain. For bone fluid calcium imbalance, the model predicts that complete turnover occurs after 261 days of formation under constant 1000 microstrain stimulus and that turnover never occurs under strain-free conditions. These predictions were not impacted by mean dynamic input strain stimuli of 1000 and 2000 microstrain at 1 Hz and 1000 microstrain amplitude. The formation phase of remodeling lasts longer than the resorption phase, increased strain stimulus accelerates bone turnover, while absence of strain significantly delays or prevents it, and formation time for turnover under monotonic increasing strain conditions is intermediate to those for constant strain stimuli at the minimum and maximum monotonic strain levels. These results are consistent with the biology, and with Frost's mechanostat theory.     

Development of the mouse mandibles and clavicles in the absence of skeletal myogenesis

In this report we employed double-knock-out mouse embryos and fetuses (designated as Myf5-/-: MyoD-/- that completely lacked striated musculature to study bone development in the absence of mechanical stimuli from the musculature and to distinguish between the effects that static loading and weight-bearing exhibit on embryonic development of skeletal system. We concentrated on development of the mandibles (= dentary) and clavicles because their formation is characterized by intramembranous and endochondral ossification via formation of secondary cartilage that is dependent on mechanical stimuli from the adjacent musculature. We employed morphometry and morphology at different embryonic stages and compared bone development in double-mutant and control embryos and fetuses. Our findings can be summarized as follows: a) the examined mutant bones had significantly altered shape and size that we described morphometrically, b) the effects of muscle absence varied depending on the bone (clavicles being more dependent than mandibles) and even within the same bone (e.g., the mandible), and c) we further supported the notion that, from the evolutionary point of view, mammalian clavicles arise under different influences from those that initiate the furcula (wishbone) in birds. Together, our data show that the development of secondary cartilage, and in turn the development of the final shape and size of the bones, is strongly influenced by mechanical cues from the skeletal musculature.     

New Developmental Evidence Clarifies the Evolution of Wrist Bones in the Dinosaur–Bird Transition

From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ossifications. Their identity is uncertain, with different labels used in palaeontology and developmental biology. We examined embryos of several species and studied chicken embryos in detail through a new technique allowing whole-mount immunofluorescence of the embryonic cartilaginous skeleton. Beyond previous controversy, we establish that the proximal–anterior ossification develops from a composite radiale+intermedium cartilage, consistent with fusion of radiale and intermedium observed in some theropod dinosaurs. Despite previous claims that the development of the distal–anterior ossification does not support the dinosaur–bird link, we found its embryonic precursor shows two distinct regions of both collagen type II and collagen type IX expression, resembling the composite semilunate bone of bird-like dinosaurs (distal carpal 1+distal carpal 2). The distal–posterior ossification develops from a cartilage referred to as “element x,” but its position corresponds to distal carpal 3. The proximal–posterior ossification is perhaps most controversial: It is labelled as the ulnare in palaeontology, but we confirm the embryonic ulnare is lost during development. Re-examination of the fossil evidence reveals the ulnare was actually absent in bird-like dinosaurs. We confirm the proximal–posterior bone is a pisiform in terms of embryonic position and its development as a sesamoid associated to a tendon. However, the pisiform is absent in bird-like dinosaurs, which are known from several articulated specimens. The combined data provide compelling evidence of a remarkable evolutionary reversal: A large, ossified pisiform re-evolved in the lineage leading to birds, after a period in which it was either absent, nonossified, or very small, consistently escaping fossil preservation. The bird wrist provides a modern example of how developmental and paleontological data illuminate each other. Based on all available data, we introduce a new nomenclature for bird wrist ossifications.     

Role of tissue interaction between pineal primordium and neighboring tissues in avian pineal morphogenesis studied by intraocular transplantation

Tissue interactions play an essential role in organogenesis during embryonic development. However, virtually no attempts have been made to study the role of tissue interaction in pineal development. In the present study we examined the inductive role of the epidermis and mesenchyme in the morphogenesis of quail pineal glands. The pineal rudiment is first observed at embryonic day 2 (E2: 2 days of incubation) at the dorsal midline of the diencephalon as a short semi-spherical protrusion. Electron microscopic observations revealed that no mesenchymal cells are found between the epidermis and the distal end of the E2 pineal primordium but that a thin layer of mesenchymal cells separate the epidermis from the pineal primordium at E3. Small pieces containing pineal rudiment were cut off from E2 or E3 embryos. They were treated with enzymes to eliminate the epidermis and/or mesenchyme, grafted into E5 chicken eyes, and cultured there for 1 week. When E3 pineal rudiment was treated with Dispase to remove the epidermis, the pineal gland developed normally. When the rudiment was further treated with collagenase to remove the surrounding mesenchymal cells, a multi-follicular structure was still formed, but to a lesser extent than when rudiments were treated with Dispase alone. When E2 quail pineal rudiment with the epidermis was grafted without any treatment, a multi-follicular structure developed which morphologically resembled embryonic pineal organs. When the epidermis was removed from E2 rudiments by Dispase, a single large vesicular structure was formed. These results suggest that the overlying epidermis and/or mesenchymal cells play some inductive role in the initial pineal development, while the mesenchymal tissue plays an important role in pineal follicular formation later during development. Since only a few experimental studies have been done to examine pineal morphogenesis, the present study provides fundamental insights into avian pineal development.