Direct Ossification Research Articles

The Impact of Aging on Bone Regeneration in Mice

Older people are projected to outnumber children for the first time in U.S. history by 2030 (U.S. Census Bureau, 2017), creating a tsunami of people with reduced bone strength who are more susceptible to bone injury and fracture. Decreased bone quality has a multivariate causal path including loss of progenitor cells, reduced anabolic and catabolic capabilities of cells, and increased senescence. The age‐related cellular decline of bone begins prior to the onset of bone diseases such as osteoporosis. Thus, aging models exclusive of disease can be immensely beneficial in finding novel angles of approach for better bone healing during aging. With this in mind, our study uses the well‐characterized mouse digit amputation model. After amputation of the digit tip young mice are able to regrow patterned bone via direct ossification and soft tissue. However, regeneration in aged mice is delayed, and the regenerated bone is architecturally inferior showing decreased trabecular thickness, increased trabecular spacing, and an attenuated distal growth of the bone. Here we explore the impact of aging on regeneration and bone architecture, introduce a new method of analysis for bone architecture and discuss the advantages to alternative analysis for bone regeneration in the digit model.Support or Funding InformationCOBRE Grant ‐ 2P20GM103629 in addition to CELT LS‐LAMP Grant

Ossification and development of vertebrae in the Balkan crested newt Triturus ivanbureschi (Salamandridae, Caudata)

Vertebral morphology, development, and evolution have been investigated for many decades, especially in the recent evo-devo era. Nevertheless, comparative data on development and ossification modes within the major tetrapod groups are scarce and frequently suffer from the use of a simplistic approach, resulting in simplistic generalizations about the formation of tetrapod vertebrae. Here, we describe the development and ossification of trunk vertebrae in Triturus ivanbureschi (Salamandridae, Caudata) and compare the results with published data on other related taxa. In so doing, we focus on the modes of ossification and development of the centrum and neural arches by analysing three developmental stages defined by the degree of limb development: stages 47, 52, and 62 according to Glücksohn (1932). Our examination of histological sections through trunk vertebrae enabled us to identify three modes of ossification within single trunk vertebrae: (i) perichordal (direct ossification of the connective tissue surrounding the notochord); (ii) perichondrial (direct ossification of the perichondrium, consisting of cartilage-covering connective tissue), and (iii) endochondral (ossification within the preformed cartilage template). We also noted the presence of intravertebral or notochordal cartilage. Although our results indicate that this cartilage develops within the notochord surrounded by the continuous notochordal sheath, more detailed further studies could shed light on its origin and development.

Vertebral Development in Paleozoic and Mesozoic Tetrapods Revealed by Paleohistological Data.

Basal tetrapods display a wide spectrum of vertebral centrum morphologies that can be used to distinguish different tetrapod groups. The vertebral types range from multipartite centra in stem-tetrapods, temnospondyls, and seymouriamorphs up to monospondylous centra in lepospondyls and have been drawn upon for reconstructing major evolutionary trends in tetrapods that are now considered textbook knowledge. Two modes of vertebral formation have been postulated: the multipartite vertebrae formed first as cartilaginous elements with subsequent ossification. The monospondylous centrum, in contrast, was formed by direct ossification without a cartilaginous precursor. This study describes centrum morphogenesis in basal tetrapods for the first time, based on bone histology. Our results show that the intercentra of the investigated stem-tetrapods consist of a small band of periosteal bone and a dense network of endochondral bone. In stereospondyl temnospondyls, high amounts of calcified cartilage are preserved in the endochondral trabeculae. Notably, the periosteal region is thickened and highly vascularized in the plagiosaurid stereospondyls. Among “microsaur” lepospondyls, the thickened periosteal region is composed of compact bone and the notochordal canal is surrounded by large cell lacunae. In nectridean lepospondyls, the periosteal region has a spongy structure with large intertrabecular spaces, whereas the endochondral region has a highly cancellous structure. Our observations indicate that regardless of whether multipartite or monospondylous, the centra of basal tetrapods display first endochondral and subsequently periosteal ossification. A high interspecific variability is observed in growth rate, organization, and initiation of periosteal ossification. Moreover, vertebral development and structure reflect different lifestyles. The bottom-dwelling Plagiosauridae increase their skeletal mass by hyperplasy of the periosteal region. In nectrideans, the skeletal mass decreases, as the microstructure is spongy and lightly built. Additionally, we observed that vertebral structure is influenced by miniaturization in some groups. The phylogenetic information that can be drawn from vertebral development, however, is limited.

Engineering Small-Scale and Scaffold-Based Bone Organs via Endochondral Ossification Using Adult Progenitor Cells.

Bone development, growth, and repair predominantly occur through the process of endochondral ossification, characterized by remodelling of cartilaginous templates. The same route efficiently supports engineering of bone marrow as a niche for hematopoietic stem cells (HSC). Here we describe a combined in vitro/in vivo system based on bone marrow-derived Mesenchymal Stem/Stromal Cells (MSC) that duplicates the hallmark cellular and molecular events of endochondral ossification during development. The model requires MSC culture with instructive molecules to generate hypertrophic cartilage tissues. The resulting constructs complete the endochondral route upon in vivo implantation, in the timeframe of up to 12 weeks. The described protocol is clearly distinct from the direct ossification approach typically used to drive MSC towards osteogenesis. Recapitulation of endochondral ossification allows modelling of stromal-HSC interactions in physiology and pathology and allows engineering processes underlying bone regeneration.

Immobilizing hydroxycholesterol with apatite on titanium surfaces to induce ossification.

BackgroundImmobilizing bioactive molecules and osteoconductive apatite on titanium implants have investigated direct ossification. In this study, hydroxycholesterol (HC) was immobilized with apatite on titanium through simply adsorption or sandwich-like coating. Three kinds of hydroxycholesterol were chosen to induce ossification: 20α-hydroxycholesterol (20α- HC), 22(S)-hydroxycholesterol (22(S)-HC) and 25-hydroxycholesterol (25-HC).The effects of HC/apatite coating on ossification abilities were evaluated in vitro and in vivo.ResultsAt 6 d, adsorbed apatite/25-HC and apatite/22(S)-HC coating exhibited some cytotoxicity, while the cell viability of apatite/20α-HC coating was similar as apatite coating. Immobilizing HC with apatite significantly enhanced the ALP activities compared with apatite coating. There was no significant difference in ALP value between adsorbed apatite/HC coating and sandwich-like apatite/HC/apatite coating. When compared with apatite coating, the mineral deposition improved by adsorbed HC with apatite at higher concentration in vivo.ConclusionsWhen compared with apatite coating, immobilizing HC with apatite coating induced the ossification in vitro and in vivo.

Endogenous bone regeneration is dependent upon a dynamic oxygen event.

Amputation of the digit tip within the terminal phalangeal bone of rodents, monkeys, and humans results in near-perfect regeneration of bone and surrounding tissues; however, amputations at a more proximal level fail to produce the same regenerative result. Digit regeneration is a coordinated, multifaceted process that incorporates signaling from bioactive growth factors both in the tissue matrix and from several different cell populations. To elucidate the mechanisms involved in bone regeneration we developed a novel multi-tissue slice-culture model that regenerates bone ex vivo via direct ossification. Our study provides an integrated multi-tissue system for bone and digit regeneration and allows us to circumvent experimental limitations that exist in vivo. We used this slice-culture model to evaluate the influence of oxygen on regenerating bone. Micro-computed tomography (µCT) and histological analysis revealed that the regenerative response of the digit is facilitated in part by a dynamic oxygen event, in which mutually exclusive high and low oxygen microenvironments exist and vacillate in a coordinated fashion during regeneration. Areas of increased oxygen are initially seen in the marrow and then surrounding areas of vasculature in the regenerating digit. Major hypoxic events are seen at 7 days postamputation (DPA 7) in the marrow and again at DPA 12 in the blastema, and manipulation of oxygen tensions during these hypoxic phases can shift the dynamics of digit regeneration. Oxygen increased to 21% oxygen tension can either accelerate or attenuate bone mineralization in a stage-specific manner in the regenerative timeline. These studies not only reveal a circumscribed frame of oxygen influence during bone regeneration, but also suggest that oxygen may be one of the primary signaling influences during regeneration.

Gene expression profiling in human craniosynostoses: a tool to investigate the molecular basis of suture ossification

Non-syndromic craniosynostoses (NSC) occur as isolated skull malformations due to the premature ossification of one (single-suture forms) or more (complex forms) calvarial sutures and represent the most frequent form of craniosynostosis worldwide. The etiology of NSC is still largely unknown as a genetic basis can be rarely demonstrated especially in single-suture forms. In these cases, during the prenatal/perinatal development of affected patients, only one suture undergoes a premature direct ossification within an otherwise physiologically grown skull. This could suggest that definite somatic alterations, possibly due to unclear environmental agents, occur locally at the site of premature suture fusion during skull development. A promising tool to investigate the molecular mechanisms that may orchestrate this event is the comparative analysis of suture- and synostosis-derived tissues and cells. This review focuses on the different studies that attempted to clarify this issue using genome-wide microarray-based technologies for the comparative analysis of gene expression profiles. All relevant results have been comprehensively reviewed, possibly compared, and critically discussed. Due to the heterogeneity of the dataset available in the literature, a univocal CRS-associated molecular profile could not be deciphered. Most differentially expressed genes are found in different studies to be involved in extracellular matrix remodeling.

BMP signaling induces digit regeneration in neonatal mice

The regenerating digit tip of mice is a novel epimorphic response in mammals that is similar to fingertip regeneration in humans. Both display restricted regenerative capabilities that are amputation-level dependent. Using this endogenous regeneration model in neonatal mice, we have found that noggin treatment inhibits regeneration, thus suggesting a bone morphogenetic protein (BMP) requirement. Using non-regenerating amputation wounds, we show that BMP7 or BMP2 can induce a regenerative response. BMP-induced regeneration involves the formation of a mammalian digit blastema. Unlike the endogenous regeneration response that involves redifferentiation by direct ossification (evolved regeneration), the BMP-induced response involves endochondral ossification (redevelopment). Our evidence suggests that BMP treatment triggers a reprogramming event that re-initiates digit tip development at the amputation wound. These studies demonstrate for the first time that the postnatal mammalian digit has latent regenerative capabilities that can be induced by growth factor treatment.

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.

Particulate Bone Allograft Incorporation in Regeneration of Osseous Defects; Importance of Particle Sizes

Packing of bone defect with particulate allografts is a commonly performed clinical procedure. However, the ideal size of bone particles used to fill bone defects is ill-defined. For this reason the study of biology of bone allografts with different particle sizes has been performed. Standard size bone defects in the femur and the tibia of experimental animals were filled with freeze-dried cortical bone allografts with particle sizes of 1-2mm, 800-500μm, 500-300μm, 300-90μm, 250-125μm, 125-106μm, 106 to 75μm and 75-25μm. Unfilled defects and those filled with autologous bone served as controls. Cortical bone was chosen because it produced better clinical results than did cancellous bone. Likewise freeze-dried particulate bone effected more rapid healing than did frozen bone. Numerical scores were assigned to each defect based on the gross, radiographic and histomorphometric studies. Particles in the range of 300 to 90 microns produced rapid healing by direct ossification. Particles below 100µm had a significantly reduced osteogenic potential. Particles in the range of 75-25μm failed to heal the defects all together. Healing of defects packed with particles larger than 300μm was slower than with 300 – 90 μm grafts. Rapid healing of bone defects packed with particulate bone allografts in the range of 300 to 90μm indicates such allografts can be used effectively in the filling of bone defects. This is of clinical relevance.

Growth plate formation and development in alligator and mouse metapodials: evolutionary and functional implications

Mammalian metapodials (metacarpals and metatarsals), unlike most long bones, form a single growth plate, and undergo longitudinal growth at only one end. The growth dynamics of non-mammalian tetrapod metapodials have not been systematically examined in order to determine if unidirectional growth is unique to mammals. Here we compare murine metapodial ossification in growth stages that parallel those of embryonic, juvenile and subadult American alligators (Alligator mississippiensis). Safranin O staining was used for qualitative histology, and chondrocyte differentiation and proliferation were assessed via immunohistochemistry for type X collagen and proliferative cell nuclear antigen (PCNA). We establish that growth plates form at both ends of alligator metapodials and are maintained in the subadult. PCNA results show that alligators and mice share common patterns of chondrocyte proliferation during growth plate formation. In addition, while alligators and mice differ initially in the degree of organization and pace of chondrocyte differentiation, these parameters are largely similar in established growth plates. However, the replacement of cartilage by bone is highly irregular throughout growth in the alligator, in contrast to the more uniform process in the mouse. These results indicate that while alligators and mammals share common mechanisms of chondrocyte regulation, they differ substantially in their processes of ossification. Phylogenetic analysis indicates that the direct ossification of one epiphysis and reliance on a single growth plate is a derived character (synapomorphy) in therian mammals and likely indicates an adaptation for erect quadrupedal gait.

The mechanically stable steam sterilization of bone grafts

Bone allografts are the standard material used in augmentative bone surgery. However, steam-sterilized bone has a low mechanical stability and limited ossification based on low strain-adapted bone remodelling. Here we describe a new technique which allows the bone to be autoclaved without losing its mechanical stability and osteoconductivity. The compression strength of the new material was compared with steam-sterilized and fresh bone based on mechanical testing using bone cylinders ( n=30/group). Allogeneic new material and fresh bone were press-fit implanted into rabbit patellar grooves and examined under fluorescent light and conventional microscopy. Initial healing was assessed after 30 d ( n=5/group). Osseous integration and remodelling was studied after 100 d ( n=12/group). Steam-sterilized bone showed no mechanical stability, whereas the new material was stiff and had compression curves similar to fresh bone; both groups showed equal degrees of direct ossification after 30 d, advanced bony ingrowth and remodelling after 100 d, and similar ingrowth depths on histomorphometric analysis. The new method preserved the stiffness and osteoconductivity of bone after steam sterilization, and microstructure, mineralization, and composition were conserved. This technique could be useful for bone banking in Third World countries.

Ossification of the mouse metatarsal: Differentiation and proliferation in the presence/absence of a defined growth plate

There is significant diversity in growth plate behavior among sites within an individual skeleton and between skeletons of different species. This variation within wild-type animals is an underutilized resource for studying skeletal development. One bone that potentially exhibits the most diverse behavior is the metatarsal. While one end forms a growth plate with an epiphyseal secondary center of ossification as in other long bones, the opposite end undergoes direct ossification in a manner more similar to short bones. Although descriptions of human metatarsal/metacarpal ossification are available, a detailed comparative analysis has yet to be conducted in an animal model amenable to biomolecular analysis. Here we report an analysis of proximal and distal ossification in an age series of mouse metatarsals. Safranin O staining was used for qualitative and quantitative histology, and chondrocyte differentiation and proliferation were analyzed using immunohistochemistry for type X collagen and proliferative cell nuclear antigen expression. We establish that, as in the human, both growth plate formation and direct ossification occur in the mouse metatarsal, with chondrocyte populations showing distinct differentiation patterns at opposite ends of the bone. In addition, growth plate formation is characterized by a peak of proliferation in reserve zone chondrocytes that distinguishes it from both established growth plates and direct ossification. Our analysis demonstrates that the mouse metatarsal is a productive model for investigating natural variation in ossification that can further understanding of vertebrate skeletal development and evolution.

BMP-2 liberated from biomimetic implant coatings induces and sustains direct ossification in an ectopic rat model

Introduction Using a rat model, we evaluated the kinetics and histomorphometry of ectopic bone formation in association with biomimetic implant coatings containing BMP-2. Materials and methods One experimental and three control groups were set up: titanium-alloy discs coated with a biomimetically co-precipitated layer of calcium phosphate and BMP-2 [1.7 μg per disc (incorporated-BMP group)]; uncoated discs (control); discs biomimetically coated with a layer of calcium phosphate alone (control); and discs biomimetically coated with a layer of calcium phosphate bearing superficially adsorbed BMP-2 [0.98 μg per disc (control)]. Discs ( n = 6 per group) were implanted subcutaneously in rats and retrieved at 7-day intervals over a period of 5 weeks for kinetic, histomorphometrical, morphological and histochemical analyses. Results In the incorporated-BMP-2 group, osteogenic activity was first observed 2 weeks after implantation and thereafter continued unabated until the end of the monitoring period. The net weekly rates of bone formation per disc were 5.8 mm 3 at 2 weeks and 3.64 mm 3 at 5 weeks. The total volumes of bone formed per disc at these junctures were 5.8 mm 3 and 10.3 mm 3, respectively. Bone tissue, which was formed by a direct ossification mechanism, was deposited at distances of up to 340 μm from the implant surfaces. The biomimetic coatings were degraded gradually, initially by foreign body giant cells alone and then also by osteoclasts. Forty percent of the coating material (and thus presumably of the incorporated BMP-2) remained at the end of the monitoring period. Hence, 60% of the incorporated BMP-2 had been released. At this 5-week juncture, no bone tissue was associated with any of the control implants. Conclusion BMP-2 incorporated into biomimetic calcium phosphate coatings is capable not only of inducing bone formation at an ectopic site in vivo but also of doing so with a very high potency at a low pharmacological level, and of sustaining this activity for a considerable period of time. The sustainment of osteogenic activity is of great clinical importance for the osseointegration of dental and orthopedic implants.

Progression of calvarial bone development requires Foxc1 regulation of Msx2 and Alx4

Calvarial bones form by direct ossification of mesenchyme. This requires condensation of mesenchymal cells which then proliferate and differentiate into osteoblasts. Congenital hydrocephalus (ch) mutant mice lack the forkhead/winged helix transcription factor Foxc1. In ch mutant mice, calvarial bones remain rudimentary at the sites of initial osteogenic condensations. In this study, we have localized the ossification defect in ch mutants to the calvarial mesenchyme, which lacks the expression of transcription factors Msx2 and Alx4. This lack of expression is associated with a reduction in the proliferation of osteoprogenitor cells. We have previously shown that BMP induces Msx2 in calvarial mesenchyme (Development 125, 1241–1251, 1998). Here, we show that BMP also induces Alx4 in this tissue. We also show that BMP-induced expression of Msx2 and Alx4 requires Foxc1. We therefore suggest that Foxc1 regulates BMP-mediated osteoprogenitor proliferation and that this regulation is required for the progression of osteogenesis beyond the initial condensations in calvarial bone development.

Contribution of single somites to the skeleton and muscles of the occipital and cervical regions in avian embryos.

Controversy has surrounded the process of resegmentation of cervico-occipital somites. We have reinvestigated this topic by grafting single somites of quail embryos homotopically into chick embryos. Somites one to five contribute to the skull. Somites one and two contribute to the parasphenoid, which develops by direct ossification in a non-segmental fashion. All cartilaginous derivatives of the somites are segmental. Somite two forms a stripe of cells in the basioccipital, exoccipital and supraoccipital. Somites three to five give rise to the subsequent caudal parts of the basioccipital and exoccipital. Somite five forms the first motion segment including the occipital condyle, the cranial part of the atlas and the tip of the dens axis. Therefore, the border between head and neck is in the centre of somite five, and corresponds to the expression boundary of Choxb-3. Somite six forms the caudal part of the atlas and the cranial part of the axis. Somites two to eight all contribute to the cranio-cervical muscles with the exception of the Mm. rectus capitis dorsalis and ventralis and the M. biventer cervicis, which do not receive contributions from somite two. In contrast, the M. cucullaris capitis is exclusively formed by myogenic cells from somite two, which parallels its exclusive innervation by the accessory nerve. Our data confirm the segmental nature of the occiput, and show that resegmentation is a very regular process involving all except the four cranialmost somites. Except for somites one and two, all of the somites contribute to the muscles located at the appropriate levels.

Biology of the calcium phosphate integration in human long bones

Calcium phosphate ceramics implanted in bones are integrated following a sequence of biological events which has already been described. We had the opportunity to analyze histologically some HA-ceramic samples that had been implanted in humans for various periods of time. Eight biopsies were obtained from calcium phosphate ceramics implanted in different sites. All the samples were implanted for periods ranging from 4 to 15 months. The samples were fixed in ethanol and embedded in hydroxyethylmetacrylate or polymethyl methacrylate. 5 μm thick sections were prepared and stained with Giemsa or by Von Kossa method. Alkaline phosphatase and tartrate resistant acid phosphatase (TRAP) were revealed by histochemical methods. Different osteointegration stages were evidenced. In early stages, hematoma remnants were still visible. These implants were invaded by a loose connective tissue in which some cell condensations were apparent. They lead to the formation of bone trabeculae by direct ossification. Some osteoblasts differentiated at the ceramic surface and synthesized osteoid tissue on the material. The fibroblast-like cells of the loose connective tissue were TRAP+. Most of them had TRAP+ nucleus and contained no cytoplasm vesicles. Multinucleated TRAP+ osteoclasts showing TRAP- negative nuclei were found in Howship's lacunae at the bone surface. Certain implants were integrated within mature bone in a later stage.

Comparative histological studies of bone and cartilage formations induced by various BMP-carrier composites.

Five different types of carriers combined with bone morphogenetic protein (BMP) were implanted into the dorsal subcutaneous tissue in the rat in order to study and compare the osteoinductive capacity and histological patterns. The carriers were: insoluble bone matrix (IBM), porous particles of hydroxyapatite (PPHAP), wet and dry collagen beads (CBw, CBd), fibrous collagen membrane (FCM), and fibrous glass membrane (FGM). The carriers alone were implanted for control studies. Each composite and its surrounding tissues were removed after 1, 2 and 3 weeks, and were studied by routine histology. Our results demonstrated that BMP-IBM, BMP-CBd, BMP-FCM and BMP-FGM induced bone tissue in a process that resembled an endochondral ossification mode; in BMP-IBM and BMP-CBd there was an early initiation of the process, in contrast with BMP-FCM and BMP-FGM where a delayed endochondral ossification was observed. These systems, with the exception of BMP-FGM, also induced bone formation through a direct ossification mode. On the other hand, BMP-PPHAP and BMP-CBw induced bone formation in a process that resembled an intramembranous ossification. BMP-carrier composites induce immature mesenchymal cells to become either osteogenetic or chondrogenetic cells, or both, depending on the physicochemical nature of the carrier that affected the microenvironment for cell differentiation. The presen study suggests that BMP-FCM, BNIP-PPHAP and BMP-CBd could be applicable carriers to explore the osteoinductive function of BMP, since no side-effects caused by the carrier were observed.

Ossification and pseudoepiphysis formation in the ?nonepiphyseal? end of bones of the hands and feet

Metacarpals, metatarsals, and phalanges were studied to assess the developmental morphology of "secondary" ossification in the "nonepiphyseal" ends of these bones as well as the formation of the pseudoepiphysis as an epiphyseal ossification variant. Both direct ossification extension from the metaphysis into the epiphysis and pseudoepiphysis formation preceded, and continued to be more mature than, formation and expansion of the "classic" epiphyseal (secondary) ossification center at the opposite end of each specific bone. Direct metaphyseal to epiphyseal ossification usually started centrally and expanded hemispherically, replacing both physeal and epiphyseal cartilage simultaneously. In contrast, when remnants of "physis" were retained, while juxtaposed epiphyseal cartilage was replaced, a pseudoepiphysis formed. There were three basic patterns of pseudoepiphysis formation. First, a central osseous bridge extended from the metaphysis across the "physis" into the epiphysis and subsequently expanded to create a mushroom-like osseous structure. In the second pattern a peripheral osseous bridge formed, creating either an osseous ring or an eccentric bridge between the metaphysis and the epiphysis. In the third pattern, multiple bridging occurred. In each situation the associated remnant "physis" lacked typical cell columns and was incapable of significantly contributing to the postnatal longitudinal growth of the involved bone. Pseudoepiphyses were well formed by 4-5 years and coalesced with the rest of the bone months of years before skeletal maturation was attained at the opposite epiphyseal end, which ossified in the typical pattern (i.e., formation of a secondary center de novo completely within the cartilaginous epiphysis). This process may also affect the development and appearance of ossification within the longitudinal epiphyseal bracket ("delta phalanx").

Nonepiphyseal ossification and pseudoepiphysis formation.

Direct ossification extending from the metaphysis into the epiphysis preceded and continued to be more mature than formation and expansion of the typical epiphyseal ossification center at the opposite end of each longitudinal bone of the hand and foot. Direct metaphyseal to epiphyseal ossification usually started centrally and expanded hemispherically, replacing both physeal and epiphyseal cartilage simultaneously. When remnants of the "physis" were retained, however, while juxtaposed epiphyseal cartilage was replaced, a pseudoepiphysis formed. Three basic patterns of pseudoepiphysis formation were evident: (a) a central osseous bridge, (b) a peripheral osseous bridge, and (c) multiple bridging. In each condition, the remnant of the "physis" lacked typical cell columns capable of contributing to the postnatal longitudinal growth of the involved bone. Pseudoepiphyses were well formed by 4-5 years and coalesced with the rest of the bone months to years before skeletal maturation was attained at the opposite epiphyseal end.