Zone of Ranvier

In the studies of physeal briding, the zone of ranvier's unobstruction was critical for normal longitudinal bone growth without angular deformities. The zone of ranvier is also placed on the longitudinal side of the bone so may be key to LSJL.  As the zone of ranvier is on the longitudinal side, it can be more easily accessed than something that's within the bone itself.  Studying how much the zone of ranvier is maintained post diaphyseal/epiphyseal fusion may be key to forming new growth plates.  If the zone of ranvier is maintained to any degree post fusion than it may be possible to restore growth.

Here's an image of the zone of ranvier:
From Normal Bone Formation.

"The growth plate may be divided anatomically into three components: a cartilaginous component, itself divided into various histologic zones; a bony component, metaphysis; and a fibrous component surrounding the periphery of the plate comprising the groove of Ranvier and the perichondrial ring of LaCroix."

"The epiphyseal artery supplies the epiphysis, or the secondary center of ossification, which itself is not part of the growth plate. Small branches arise at right angles to the main epiphyseal artery in the epiphysis and pass through small cartilage canals in the reserve zone to terminate at the top of the cell columns in the proliferative zone. Each small branch from the epiphyseal artery arborizes in rakelike fashion to supply the top portion of from four to ten cell columns. The proliferative zone, therefore, is well supplied with blood. None of the branches from the epiphyseal arteries penetrate the cartilage portion of the growth plate beyond the uppermost part of the proliferative zone; that is, no vessels pass through the proliferative zone to supply the hypertrophic zone."

"The reserve zone lies immediately adjacent to the secondary bony epiphysis. Various terms have been applied to this zone, including resting zone, zone of small-size cartilage cells, and germinal zone. However, these cells are not resting, are not small in comparison with the cells in the proliferative zone, and they are not germinal cells. They appear to store lipid and other materials and perhaps are held in reserve for later nutritional requirements. If that is true, the term reserve zone may be appropriate. The cells in this zone are spherical, exist singly or in pairs, are relatively few when compared with the number of cells in other zones, and are separated from each other by more extracellular matrix than are cells in any other zone. The cells in the reserve zone are approximately the same size as the cells in the proliferative zone. The cytoplasm exhibits a positive staining reaction for glycogen.  These cells contain abundant endoplasmic reticulum, a clear indication that they are actively synthesizing protein. They contain more lipid bodies and vacuoles than do cells in other zones but contain less glucose-6-phosphate dehydrogenase, lactic dehydrogenase, malic dehydrogenase, and phosphoglucoisomerase. The zone also contains the lowest amount of alkaline and acid phosphatase, total and inorganic phosphate, calcium, chloride, potassium, and magnesium. The matrix in the reserve zone contains less lipid, glycosaminoglycan, protein polysaccharide, moisture, and ash than the matrix in any other zone. It exhibits less incorporation of radiosulfur (35S) than any other zone and also shows less Iysozyme activity than the other zones. It contains the highest content of hydroxyproline of any zone in the plate. Collagen fibrils in the matrix exhibit random distribution and orientation. Matrix vesicles are also seen in the matrix, but they are fewer than in other zones. The matrix shows a positive histochemical reaction for the presence of a neutral mucopolysaccharide or an aggregated proteoglycan."

Another image of the Zone of Ranvier and LaCroix:


"The perichondrial ring is a dense fibrous band that encircles the growth plate at the bone-cartilage junction and in which collagen fibers run vertically, obliquely, and circumferentially. It is continuous at one end with the group of fibroblasts and collagen fibers in the ossification groove and at the other end with the periosteum and subperiosteal bone of the metaphysis. In rodents, rabbits, and dogs, the innermost layer of the perichondrial ring consists of bone that may or may not be attached to the subperiosteal bone of the metaphysis. This cylindrical sheath of bone may not be present in all species at all ages in all growth plates. For instance, it is not present in the proximal femur in the human at any age. " Whether or not bone is present in the perichondrial ring, there is no doubt that the ring provides mechanical support for the otherwise weak bone-cartilage junction of the growth plate"

If we can somehow prove that the zone of Ranvier is retained post fusion then that would be a huge breakthrough for forming new growth plates.

Identification of a stem cell niche in the zone of Ranvier within the knee joint.

"A superficial lesion of the articular cartilage does not spontaneously self-repair and has been suggested to be partly due to lack of progenitor cells within the joint that can reach the site of injury. To study whether progenitor cells are present within the joint, 3-month-old New Zealand white rabbits were exposed to bromodeoxyuridine (BrdU) for 12 consecutive days and were then sacrificed 4, 6, 10, 14, 28 and 56 days after the first BrdU administration. Presence of BrdU and localization of progenitor markers were detected. After 10 days of BrdU exposure, BrdU-positive cells, i.e. proliferating cells, were abundantly detected in the epiphyseal plate, the perichondrial groove of Ranvier, and in all zones of the articular cartilage{so the rabbits were skeltally immature}. After a wash-out period, BrdU-positive cells were still present, i.e. those considered to be progenitor cells, in these regions of the knee except for the proliferative zone of the epiphyseal plate. Cells in the perichondrial groove of Ranvier were further positive for several markers associated with progenitor cells and stem cell niches, including Stro-1, Jagged1, and BMPr1a. A small population of progenitor cells is present in the perichondrial groove of Ranvier as well as within the articular cartilage in the knee. The perichondrial groove of Ranvier demonstrates the properties of a stem cell niche."

"The growth plate is surrounded by an encircling fibrochondrosseous structure. This anatomical structure consists of the zone of Ranvier and the ring of LaCroix. The area [harbors] prechondrocytes responsible for the circumferential growth of cartilage."

"perichondrial cells from the ring of LaCroix, which is a fibrous band that surrounds the groove of Ranvier and is continuous with the periosteum of the metaphysis, serve as a reservoir for precartilaginous cells in the germinal layer of the epiphyseal growth plate"

"A larger number of BrdU-positive cells in the epiphyseal plate[were] near the perichondrial groove of Ranvier than in the central area of the epiphyseal plate. [Cells may migrate] from the perichondrial groove of Ranvier into the epiphysis."<-Based on how much the Zone of Ranvier is retained this can be used to form new growth plates.

Regulation of rapid signal transducer and activator of transcription-5 phosphorylation in the resting cells of the growth plate and in the liver by growth hormone and feeding.

"The Janus kinase (Jak)-signal transducer and activator of transcription (STAT)-5 pathway is activated by GH, so we developed a method to visualize nuclear Stat5b and phosphorylated Stat5 in single cells in response to a pulse of GH. Hep2 cells did not show a Stat5 phosphorylation (pY-Stat5) response to GH except in cells transfected to express GH receptors. ATDC5 cells express GH receptors and showed GH-induced pY-Stat5 responses, which varied with their state of chondrocyte differentiation. In vivo, Stat5b(+ve) nuclei were seen in the resting and prehypertrophic chondrocytes of the growth plate. After a single ip pulse of human GH or mouse GH, but not prolactin, pY-Stat5 responses were visible in cells in the resting zone and groove of Ranvier, 10-45 min later{Maybe LSJL mimics these pulses}. Prehypertrophic chondrocytes showed no pY-Stat5 response to GH. GH target cells were also identified in other tissues, and a marked variability in spatiotemporal pY-Stat5 responses was evident. Endogenous hepatic pY-Stat5 was detected in mice with intact GH secretion but only during a GH pulse. Fasting and chronic exposure to GH attenuated the pY-Stat5 response to an acute GH injection.  pY-Stat5 responses to GH vary in time and space, are sensitive to nutritional status, and may be inhibited by prior GH exposure{GH needs to be cycled}. GH [regulates] the fate of immature chondrocytes."

"Although Stat1, -3, -5a, and -5b can all be activated by GH, Stat5b is the major target for growth promotion because it is uniquely responsive to the temporal pattern of plasma GH"

Groove of Ranvier identified in A.

Development of the distal femoral epiphysis: a microscopic morphological investigation of the zone of Ranvier.

"The distal femoral epiphysis, physis, and contiguous metaphysis were examined radiographically, morphologically, and histologically in 97 human specimens ranging in age from 9 prenatal weeks to 16 postnatal years. The earliest development of the femoral anlage was characterized by patterns of appositional and interstitial chondrogenesis throughout its entire structure. Once central endochondral ossification began, chondrogenic interstitial and appositional growth became regionally restricted to the femoral epiphyses. Interstitial chondrogenesis became limited to the germinal region of the developing physis, and appositional chondrogenesis was restricted to the region of loosely packed cells of the perichondrial ossification zone of Ranvier. Appositional chondrogenesis within the perichondrium appears to make its greatest contribution to transverse expansion of the distal femoral epiphysis during the first 5 months of gestation. After the sixth month of gestation, the perichondrial appositional growth contribution appears to decline steadily."

Appositional growth by deposition underneath the periosteum and interstitial growth are two methods suggested by the study for growth of the physis in width(it doesn't say what the mechanism of interstitial growth is).

Eventually the zone of ranvier is not apparent.  The bone collar incorporates into the metaphyseal bone.  There is a region of loosely packed cells that does seem to persist.

According to The expression of the nuclear oncogenes c-myc and c-jun in the groove of Ranvier of the rabbit growth plate., cells in the Groove of Ranvier are positive for c-Myc and c-Jun.

Role of the ossification groove of Ranvier in normal and pathologic bone growth: a review.

"cells in the groove [of Ranvier] and adjacent periosteum contain type II collagen messenger RNA (mRNA) characteristic of cartilage"

"The normal interstitial growth of the reserve cell zone or the germinal layer of the growth plate leads to migration of cells toward the periphery, where some cells give rise to new cell columns inside the groove. Other cells have their structure changed and enter the inner cell layer of the well-vascularized tissue in the ossiffication groove. These cells lose their surrounding ground substance entering the groove but retain their ability to synthesize type II collagen mRNA. With continued growth of the bone, the cells are left behind and give rise to osteoblasts and bone."

 The periphysis and its effect on the metaphysis: I. Definition and normal radiographic pattern

"The zone of Ranvier and the ring of LaCroix, together with the membranous bone bark they produce, are termed the periphysis in order to emphasize their normal effect (the metaphyseal collar) on the metaphysis of the infant and young child. In the first 7 years of life, the normal collar at the wrist is 1-3 mm wide. The step-off between the metaphyseal collar and the curvilinear metaphysis, at the margin of the periphysis, should not be mistaken for abuse fracture. The periphyseal bone bark may be radiologically visible at the edge of the physis at the distal ulna in 9% of infants and should not be mistaken for fracture or rickets." 

"The periphysis surrounds the growth plate (physis) of tubular bones and also the most recently formed several millimeters of metaphysis in infants. It is a fibrochondroosseous structure that (a) appears to maintain the transverse diameter of the physis and at the same time (b) allows gradual transverse growth of the same physis. That portion of the periphysis adjacent to the physis has been described under the names zone or groove of Ranvier, that portion adjacent to the metaphysis, as the
ring of LaCroix.
Histologically, the Ranvier and LaCroix zones are a single structure; both lay down a continuous thin layer of bone, termed bone bark, centrally at the periphery of the physis and metaphysis. This bone bark is produced by membranous, rather than enchondral, bone formation. In the first several years of life that portion of the metaphysis surrounded by the periphysis has a flat, longitudinally directed periphery on radiographs, rather than a smooth curved contour characteristic of the margins of other portions of the metaphysis. The result is the short metaphyseal collar"

"The periphysis [restrains] longitudinal widening of the physis."

Cartilage Tissue Engineering; the search for chondrogenic progenitor cells and associated signalling pathways

"Stem cells [are] not only in the articular cartilage but also in the groove of Ranvier located in the periphery of the epiphyseal growth plate.
The groove of Ranvier exhibited properties as a stem cell niche structure. Further biopsies
from human normal articular cartilage, as well as regenerated and repaired cartilage after ACI
were studied. The human normal articular cartilage demonstrated expression of the stem cell
associated markers STRO-1 and Bcrp1 in cells in the superficial zone, and activity of the
fundamental Wnt (Wingless-related proteins) and Notch signalling pathways. The distribution
showed a distinct zonal pattern in the normal cartilage. In biopsies from regenerated cartilage
with almost normal histological architecture, the markers and pathways studied demonstrated a
distinct zonal pattern similar to that in normal cartilage."

"in articular cartilage there are subpopulations of cells with mesenchymal stem cell properties"

"From the lateral plate mesoderm, undifferentiated mesenchymal cells begin to migrate to areas destined to become bone, followed by tight packing of the cells, known as mesenchymal condensation. The cartilage anlagen for the future skeletal elements have now formed. Cellular condensation is associated with increased cell to cell contact and increased cell to matrix interaction. Molecules taking part in the intercellular communication are e.g. neural cell adhesion molecule (N-CAM), Ncadherin, tenascin, versican, fibronectin and gap junctions (connexin 42 and 43),"

"The first sign of joint formation is the appearance of an interzone. The interzone cells gives rise to the articular layer of the future long bones while the chondrocytes developing from the mesenchymal condensation are assumed to be a part of epiphyseal growth plate and to take part in endochondral ossification, these cells are called transient chondrocytes. It has been unclear whether the interzone cells derive from transdifferentiation of local prechondrocytes into interzone cells or if there is migration of mesenchymal cells into the joint site, or a combination"

"It[perichondrial groove of Raniver] is a circumferential anatomical structure in the periphery of the epiphyseal growth plate and consists of the zone of Ranvier and the ring of LaCroix. It is a well defined structure in the growing skeleton. In the adult it is assumed to be integrated with the periosteum however, this has not been well explored in the adult human being."

"Markers associated with and suggested to define possible stem cells or progenitor cells in mesenchymal tissue and also, in some cases, in adult cartilage are CD105(Endoglin), CD166 (Alcam) and FGFR3 (Fibroblast Growth Factor receptor 3)"

ID1 and ID3 are involved in the proliferation of adult articular chondrocytes.

"A significant decrease in DNA synthesis was noticed when antisense nucleotides against Id1 and Id3 were added, both in normal chondrocytes and chondrosarcoma cells."

"Progenitor cells exist in the perichondrial groove of Ranvier and in the articular cartilage of rabbits (IV)"

"The markers associated with stem cells/progenitor cells and stem cell niches: Stro-1, Notch1, Patched, Jagged1, BMPr1a, 1-Integrin and N-cadherin"

"Progenitor cells exist in the knee of sexually mature rabbits and are mainly located to the perichondrial groove of Ranvier. Progenitor cells have also been detected in small numbers dispersed throughout the articular cartilage."

"The groove of Ranvier in the joint is a potential stem cell niche"

Possible Contribution of Wnt-Responsive Chondroprogenitors to the Postnatal Murine Growth Plate

"Active cell proliferation and turnover in the growth plate is essential for embryonic and postnatal bone growth. We performed a lineage tracing of Wnt/β-catenin signaling responsive cells (Wnt-responsive cells) using Axin2CreERT2;Rosa26ZsGreen mice and found a novel cell population that resides in the outermost layer of the growth plate facing the Ranvier's groove (RG; the perichondrium adjacent to growth plate). These Wnt-responsive cells rapidly expanded and contributed to formation of the outer growth plate from the neonatal to the growing stage but stopped expanding at the young adult stage when bone longitudinal growth ceases.{since these cells are WNT responsive things that stimulate WNT would stimulate longitudinal bone growth}. In addition, a second Wnt-responsive sporadic cell population was localized within the resting zone of the central part of the growth plate during the postnatal growth phase. While it induced ectopic chondrogenesis in the RG, ablation of β-catenin in the Wnt-responsive cells strongly inhibited expansion of their descendants toward the growth plate. These findings indicate that the Wnt-responsive cell population in the outermost layer of the growth plate is a unique cell source of chondroprogenitors involving lateral growth of the growth plate and suggest that Wnt/β-catenin signaling regulates function of skeletal progenitors in a site- and stage-specific manner."

"Matrix production and accumulation are also important for interstitial growth."

"During long bone growth, chondroprogenitors for the growth plate may reside in several locations. Cell labeling studies using tritiated thymidine or bromodeoxyuridine have suggested that the long-term labeled cells (slow-cycling cells), one of the characteristics of stem cells, are present in the perichondrium adjacent to the growth plate and the border between epiphyseal bone and the growth plate during postnatal growth in rodents and rabbits.The former region is called Ranvier's groove (RG) or the groove of Ranvier and the latter is the resting (reservoir) zone of growth plate. Previous histological and functional studies have addressed that these two regions likely provide new cells to the growth plate."

"Wnt proteins, likely mediated by β-catenin signaling, maintain embryonic stem cell phenotype in culture."

"During the growth plate development, Wnt/β-catenin signaling is indispensable. Inactivation of β-catenin signaling strongly affects cartilage development and induces morphological and functional abnormality of the growth plate, suggesting that Wnt/β-catenin signaling may participate in regulation of chondroprogenitors in the growth plate. We hypothesized that chondroprogenitors may be Wnt-regulated cells and can be visualized as Wnt/β-catenin signaling–responsive cells (Wnt-responsive cells)."

"This study demonstrates that a small number of specific cells in the outermost layer of the growth plate near the RG contribute to the appositional growth of the growth plate from the early postnatal to the growing stage in mice."

"decreases in number and proliferation activity of the resting chondrocytes are closely linked to reduction in bone growth with age and pathological growth arrest, indicating that the resting zone contains chondroprogenitors"