Vital Roles of ß-catenin in Trans-differentiation of Chondrocytes to Bone Cells.

A recent breakthrough showing that direct trans-differentiation of chondrocytes into bone cells commonly occurs during endochondral bone formation in the growth plate, articular cartilage, and mandibular condylar cartilage suggests that chondrogenesis and osteogenesis are likely one continuous biological process instead of two separate processes. Yet, gene regulation of this cell transformation is largely unclear. Here, we employed cartilage-specific ß-catenin loss-of-function (ß-catenin fx/fx ) and gain-of-function (ß-catenin fx(exon3)/ fx(exon3) ) models in the R26RTomato background (for better tracing the cell fate of chondrocytes) to study the role of ß-catenin in cell trans-differentiation. Using histological, immunohistochemical, and radiological methods combined with cell lineage tracing techniques, we showed that deletion of ß-catenin by either Acan-CreERT2 or Col10a1-Cre resulted in greatly reduced cell trans-differentiation with a significant decrease in subchondral bone volume during mandibular condylar growth. Molecular studies demonstrated severe defects in cell proliferation and differentiation in both chondrocytes and bone cells. The gain of function studies (constitutive activation of ß-catenin with Acan-CreERT2 at ages of postnatal day 7, 4-weeks and 6-months) led to more bone cell trans-differentiation of chondrocytes in the mandibular condyle due to increased proliferation and accelerated chondrocyte differentiation with incipient osteogenic changes within the cartilage matrix, resulting in an increased volume of poorly-formed immature subchondral bone. These results support the notion that chondrogenesis and osteogenesis are one continuous process, in which ß-catenin signaling plays an essential role in the cell trans-differentiation of chondrocytes into bone cells during mandibular condylar development and growth.

Chondrogenesis and osteogenesis are one continuous developmental and lineage defined biological process.

Although chondrogenesis and osteogenesis are considered as two separate processes during endochondral bone formation after birth, recent studies have demonstrated the direct cell transformation from chondrocytes into bone cells in postnatal bone growth. Here we use cell lineage tracing and multiple in vivo approaches to study the role of Bmpr1a in endochondrogenesis. Our data showed profound changes in skeletal shape, size and structure when Bmpr1a was deleted using Aggrecan-Cre ERT2 in early cartilage cells with a one-time tamoxifen injection. We observed the absence of lineage progression of chondrocyte-derived bone cells to form osteoblasts and osteocytes in metaphyses. Furthermore, we demonstrated the key contribution of growth plate chondrocytes and articular chondrocytes, not only for long bone growth, but also for bone remodeling. In contrast, deleting Bmpr1a in early osteoblasts with 3.6 Col 1-Cre had little impact on skeletal shape and size except for a sharp increase in osteoblasts and osteocytes, leading to a profound increase in bone volume. We conclude that chondrogenesis and osteogenesis are one continuous developmental and lineage-defined biological process, in which Bmpr1a signaling in chondrocytes is necessary for the formation of a pool or niche of osteoprogenitors that then contributes in a major way to overall bone formation and growth.

A novel auditory ossicles membrane and the development of conductive hearing loss in Dmp1-null mice.

Genetic mouse models are widely used for understanding human diseases but we know much less about the anatomical structure of the auditory ossicles in the mouse than we do about human ossicles. Furthermore, current studies have mainly focused on disease conditions such as osteomalacia and rickets in patients with hypophosphatemia rickets, although the reason that these patients develop late-onset hearing loss is unknown. In this study, we first analyzed Dmp1 lac Z knock-in auditory ossicles (in which the blue reporter is used to trace DMP1 expression in osteocytes) using X-gal staining and discovered a novel bony membrane surrounding the mouse malleus. This finding was further confirmed by 3-D micro-CT, X-ray, and alizarin red stained images. We speculate that this unique structure amplifies and facilitates sound wave transmissions in two ways: increasing the contact surface between the eardrum and malleus and accelerating the sound transmission due to its mineral content. Next, we documented a progressive deterioration in the Dmp1-null auditory ossicle structures using multiple imaging techniques. The auditory brainstem response test demonstrated a conductive hearing loss in the adult Dmp1-null mice. This finding may help to explain in part why patients with DMP1 mutations develop late-onset hearing loss, and supports the critical role of DMP1 in maintaining the integrity of the auditory ossicles and its bony membrane.

Co-localization of Cell Lineage Markers and the Tomato Signal.

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes. On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

Genetic Influences on Temporomandibular Joint Development and Growth.

The temporomandibular joint (TMJ) is a small synovial joint at which the mandible articulates with the skull during movements involved in speaking and mastication. However, the secondary cartilage lining its joint surfaces is indicative of a very different developmental history than limb cartilages. This review summarizes our current knowledge of genes that regulate the formation of primary components of the TMJ, as well as genes that regulate postnatal growth of the TMJ. Although the TMJ is regulated by some of the same genes that are important in limb joints, others appear unique to the TMJ or have different actions. Runx2, Sox9, and members of the TGF-ß/BMP family are critical drivers of chondrogenesis during condylar cartilage morphogenesis, and Indian hedgehog (Ihh) is important for formation of the articular disc and cavitation. Osterix (Osx) is a critical regulator of endochondral bone formation during postnatal TMJ growth.

Bmpr1a Signaling in Cartilage Development and Endochondral Bone Formation.

The type IA bone morphogenetic protein receptor (Bmpr1a), encoded by 11 exons and spanning about 40 kb on chromosome 14 in mice and chromosome 10 in human (Derynck & Feng, 1997; Mishina, Hanks, Miura, Tallquist, & Behringer, 2002), is an essential receptor for BMP signaling. This chapter focuses on the current understanding of the role of Bmpr1a in cartilage development and endochondral ossification, including formation of the mesenchymal condensation, chondrocyte differentiation and maturation, and endochondral bone development.

The winds of change revisited: progress towards building a culture of evidence-based dentistry.

In 2008, Texas A&M University Baylor College of Dentistry launched a comprehensive four-year curriculum in evidence-based dentistry (EBD) along with a series of faculty development initiatives to create an EBD culture. The aim of this study was to determine the institution's success in achieving this goal. The assessment tool used was the PEAK instrument, which measures respondents' EBD Practices, Experience, Attitudes, and Knowledge. Two EBD-trained classes of students and one class untrained in EBD (approximately 100 students in each class) were assessed annually. The faculty were assessed before and after completion of the initiative. Nearly all students responded, with samples ranging from 87 to 102; the faculty response rates were 53% (62/117) in 2009 and 66% in 2013 (81/123). In the results, the trained students scored significantly higher in knowledge than the untrained students at each of the first three PEAK administrations (p≤0.001). Regarding confidence in appraising a research report, the first trained group significantly gained in appropriate use of statistical tests (p<0.001), while the second trained group significantly gained in this aspect and five others (p≤0.032). At the final PEAK administration, the second trained group agreed more than the untrained group that EBD was important for the practice of dentistry (p<0.001). Faculty comfort level with reading peer-reviewed articles increased significantly from 2009 to 2013 (p=0.039). Faculty members who participated in the summer EBD Fundamentals course (n=28) had significantly higher EBD knowledge scores than those who did not participate (p=0.013), and their EBD attitudes and practices were more positive (p<0.05). Students and faculty trained in EBD were more knowledgeable and exhibited more positive attitudes, supporting a conclusion that the college has made substantial progress towards achieving an EBD culture.

Osterix controls cementoblast differentiation through downregulation of Wnt-signaling via enhancing DKK1 expression.

Osterix (Osx), a transcriptional factor essential for osteogenesis, is also critical for in vivo cellular cementum formation. However, the molecular mechanism by which Osx regulates cementoblasts is largely unknown. In this study, we initially demonstrated that overexpression of Osx in a cementoblast cell line upregulated the expression of markers vital to cementogenesis such as osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP) at both mRNA and protein levels, and enhanced alkaline phosphatase (ALP) activity. Unexpectedly, we demonstrated a sharp increase in the expression of DKK1 (a potent canonical Wnt antagonist), and a great reduction in protein levels of ß-catenin and its nuclear translocation by overexpression of Osx. Further, transient transfection of Osx reduced protein levels of TCF1 (a target transcription factor of ß-catenin), which were partially reversed by an addition of DKK1. We also demonstrated that activation of canonical Wnt signaling by LiCl or Wnt3a significantly enhanced levels of TCF1 and suppressed the expression of OPN, OCN, and BSP, as well as ALP activity and formation of extracellular mineralized nodules. Importantly, we confirmed that there were a sharp reduction in DKK1 and a concurrent increase in ß-catenin in Osx cKO mice (crossing between the Osx loxP and 2.3 Col 1-Cre lines), in agreement with the in vitro data. Thus, we conclude that the key role of Osx in control of cementoblast proliferation and differentiation is to maintain a low level of Wnt-ß-catenin via direct up-regulation of DKK1.

Critical role of Bmpr1a in mandibular condyle growth.

The importance of Bone Morphogenetic Proteins (BMPs) in the regulation of cell fate, differentiation and proliferation in the growth plate is well-known. However, in secondary cartilages (such as that in the temporomandibular joint) that grow by proliferation of prechondrocytes and differ in their pattern of growth, the role of BMPs is largely unexplored. To examine this question, we ablated Bmpr1a in the condylar cartilage of neonatal mice and assessed the consequences for mandibular condyle growth and organization at intervals over the ensuing 4 weeks. Bmpr1a deficiency caused significant chondrodysplasia and almost eliminated the chondrocytic phenotype in the TMJ. Expression of Sox9, collagen II, proteoglycan were all greatly reduced, and cell proliferation as detected by BrdU was almost non-existent in the knockout mice. Primary bone spongiosa formation was also disturbed and was accompanied by reduced Osterix expression. These findings strongly suggest that Bmpr1a is critical for the development and growth of the mandibular condyle via its effect on proliferation of prechondroblasts and chondrocyte differentiation.

Roles of notch signalling in mandibular condylar cartilage.

IMPORTANCE: Notch proteins are cell surface transmembrane spanning receptors which mediate critically important cellular functions through direct cell-cell contact. Interactions between Notch receptors and their ligands regulate cell fate decisions such differentiation, proliferation and apoptosis in numerous tissues. We have previously shown using immunohistochemistry that Notch1 is localized primarily to the prechondroblastic (chondroprogenitor) layer of the mandibular condylar cartilage (MCC). OBJECTIVE: To test if Notch signalling changes patterns of proliferation and differentiation in the MCC and to investigate if Notch signalling acts downstream of Fibroblast Growth Factor 2 (FGF-2). METHODS: Condylar cartilage explants were cultured over serum-free DMEM containing either 0 or 50nM DAPT, a Notch signal inhibitor. Explants were used for RNA extraction and immunohistochemistry. RESULTS: Analysis of gene array data demonstrated that the perichondrial layer of the MCC is rich in Notch receptors (Notch 3 and 4) and Notch ligands (Jagged and Delta) as well as various downstream facilitators of Notch signalling. Disruption of Notch signalling in MCC explants decreased proliferation (Cyclin B1 expression) and increased chondrocyte differentiation (Sox9 expression). Moreover, we found that the actions of FGF-2 in MCC are mediated in part by Notch signalling. CONCLUSION: These data suggest that Notch signalling contributes to the regulation of proliferation and differentiation in the MCC.

Genes that regulate morphogenesis and growth of the temporomandibular joint: a review.

Compared with the joints of the limbs, our understanding of the genes that regulate development and growth in the temporomandibular joint (TMJ) is fairly limited. Because the morphogenesis of the secondary cartilage and other intra-articular structures in the TMJ occurs later and in a different manner than in the limbs, the genetic control of TMJ development might reasonably be assumed to differ from that in the limbs. However, studies of the specific genes regulating TMJ morphogenesis and growth have only begun to appear in the literature within the last decade. This review attempts to survey and interpret the existing knowledge on this topic and to suggest fruitful avenues of investigation for the future. Studies to date using knockout and over-expression of candidate genes suggest that a developmental hierarchy of joint structures exists, with condyle development primary. A hierarchy of gene expression also exists: Runx2 and Sox9 expression is critical for condylar cartilage formation. Several of the other genes discussed in this report may regulate TMJ morphogenesis by affecting Sox9 and Runx2 expression and control the ihh-PTHrP axis by means of these genes.

Sarcoidosis and Sjögren's syndrome: clinical and salivary evaluation.

BACKGROUND: Sarcoidosis and Sjögren's syndrome are two different diseases; however, when affecting the salivary glands, both diseases exhibit similar clinical signs and symptoms, which often complicates the diagnosis. The purpose of this study was to investigate the possibility of using salivary electrophoresis to differentiate between the two diseases. METHODS: Saliva was collected from patients with sarcoidosis and patients with Sjögren's syndrome. Salivary flow rate, total protein, and electrophoretic profiles were examined. RESULTS: Mean salivary flow rate was 0.41 ± 0.07 ml/min/gland vs. 0.43 ± 0.07 ml/min/gland; total salivary protein was 130.0 ± 29.2 mg% vs. 104.0 ± 8.8 mg% for sarcoidosis vs. Sjögren's syndrome, respectively. No differences were observed in salivary flow rate, total salivary protein, or electrophoretic profile between patients with sarcoidosis and patients with Sjögren's syndrome (P = 0.768, 0.718, and 1.000, respectively). CONCLUSIONS: Salivary protein electrophoresis does not appear to be useful to differentiate between sarcoidosis and Sjögren's syndrome.

The evidence-based dentistry initiative at Baylor College of Dentistry.

This report describes the impact of an R25 Oral Health Research Education Grant awarded to the Texas A&M Health Science Center-Baylor College of Dentistry (BCD) to promote the application of basic and clinical research findings to clinical training and encourage students to pursue careers in oral health research. At Baylor, the R25 grant supports a multi-pronged initiative that employs clinical research as a vehicle for acquainting both students and faculty with the tools of evidence-based dentistry (EBD). New coursework and experiences in all 4 years of the curriculum plus a variety of faculty development offerings are being used to achieve this goal. Progress on these fronts is reflected in a nascent "EBD culture" characterized by increasing participation and buy-in by students and faculty. The production of a new generation of dental graduates equipped with the EBD skill set as well as a growing nucleus of faculty who can model the importance of evidence-based practice is of paramount importance for the future of dentistry.

Creating an evidence-based dentistry culture at Baylor College of Dentistry: the winds of change.

In the early years of the new millennium, the National Institute of Dental and Craniofacial Research of the National Institutes of Health began funding Oral Health Research Education Grants using the R25 mechanism to promote the application of basic and clinical research findings to clinical training and to encourage students to pursue careers in oral health research. This report describes the impact of an R25 grant awarded to the Texas A&M Health Science Center's Baylor College of Dentistry (BCD) on its curriculum and faculty development efforts. At BCD, the R25 grant supports a multipronged initiative that employs clinical research as a vehicle for acquainting both students and faculty with the tools of evidence-based dentistry (EBD). New coursework and experiences in all four years of the curriculum plus a variety of faculty development offerings are being used to achieve this goal. Progress on these fronts is reflected in a nascent EBD culture characterized by increasing participation and buy-in by students and faculty. The production of a new generation of dental graduates equipped with the EBD skill set as well as a growing nucleus of faculty members who can model the importance of evidence-based practice is of paramount importance for the future of dentistry.

Cell fate mediators Notch and Twist in mouse mandibular condylar cartilage.

OBJECTIVE: The objectives of this study were to examine if Twist and Notch 1 are present in the mandibular condylar cartilage (MCC) and whether their gene expression can be altered by exogenous FGF-2 and TGF-ß2. DESIGN: Half-heads from CD-1 mice pups harvested at embryonic day 17 (E17) were fixed, decalcified, and sectioned in the sagittal plane for immunohistochemical detection of Notch and Twist using confocal microscopy. Other mandibular condyles and adjacent ramus from E17 mice were cultured in serum-free DMEM containing 0, 3, or 30 ng/mL of FGF-2 (10-12 condyles per treatment group). This experimental design was repeated with medium containing 0, 3, or 30 ng/mL of TGF-ß2. After 3 days of culture, the pooled RNA from each group was extracted for examination of Notch and Twist gene expression using quantitative real-time RT-PCR. RESULTS: Immunohistochemical examination revealed that Notch and Twist were localized to the prechondroblastic and upper chondroblastic layers of the cartilage. Exogenous FGF-2 up-regulated Notch 1, Twist 1 and Twist 2 gene expression in MCC explants from E17 mice, whilst TGF-ß2 had the opposite effect. CONCLUSIONS: The gene expression data demonstrate that MCC explants are sensitive to growth factors known to affect Notch and Twist in other tissues. The subset of cells in which Twist and Notch immunoreactivity was found is suggestive of a role for FGF-2 and TGF-ß2 as regulators of cell differentiation of the bipotent MCC cell population, consistent with the role of Notch and Twist as downstream mediators of these growth factors in other tissues.

Distribution of small integrin-binding ligand, N-linked glycoproteins (SIBLING) in the articular cartilage of the rat femoral head.

The small integrin-binding ligand, N-linked glycoprotein (SIBLING) family is closely related to osteogenesis. Until recently, little was known about their existence in articular cartilage. In this study, we systematically evaluated the presence and distribution of four SIBLING family members in rat femoral head cartilage: dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), osteopontin (OPN), and dentin sialophosphoprotein (DSPP). First, non-collagenous proteins were extracted and then separated by ion-exchange chromatography. Next, the protein extracts eluted by chromatography were analyzed by Stains-all staining and Western immunoblotting. IHC was used to assess the distribution of these four SIBLING family members in the femoral head cartilage. Both approaches showed that all the four SIBLING family members are expressed in the femoral head cartilage. IHC showed that SIBLING members are distributed in various locations throughout the articular cartilage. The NH2-terminal fragments of DMP1, BSP, and OPN are present in the cells and in the extracellular matrix, whereas the COOH-terminal fragment of DMP1 and the NH2-terminal fragment of DSPP are primarily intracellularly localized in the chondrocytes. The presence of the SIBLING family members in the rat femoral head cartilage suggests that they may play important roles in chondrogenesis.

E-teaching and learning preferences of dental and dental hygiene students.

This project was conducted to identify student preferences for e-teaching and learning. An online Student Preferences for Learning with E-Technology Survey was developed to assess computer experiences, the use and effectiveness of e-resources, preferences for various environments, need for standardization, and preferred modes of communication. The survey was administered in May 2008 to all dental and dental hygiene students at Baylor College of Dentistry. There was an 85 percent response rate (n=366/432). About two-thirds of the students found college e-resources effective for learning. They preferred printed text over digital (64 percent) and wanted e-materials to supplement but not replace lectures (74 percent). They reported e-materials would "extensively" enhance learning, such as e-lectures (59 percent), clinical videos (54 percent), and podcasts (45 percent). They reported the need for a central location for e-resources (98 percent) and an e-syllabus for every course (86 percent) in a standard format (77 percent). One difficulty reported was accessing e-materials from external locations (33 percent). Students commented on the need for faculty training and standardization of grade posting. A qualitative theme was that e-resources should not replace interactions with faculty. Some infrastructure problems have been corrected. Planning has begun for standardization and expansion of e-resources. These improvements should enhance learning and increase the options for individualizing instruction, study strategies, and course remediation.

Timing effects of growth hormone supplementation on rat craniofacial growth.

Growth hormone (GH) supplementation has become a popular treatment approach for GH normal children with short stature. To investigate how the timing of GH supplementation affects the growth of the craniofacial region, three groups of GH-normal, 28-day-old female Wistar rats were examined over 4 weeks: the early group (n = 10) received two daily injections of rhGH (2 mg/kg/day) from days 1 to 28, the late group (n = 10) received two daily saline injections from days 1 to 14 (Phase I) followed by two daily injections of rhGH from days 14 to 28 (Phase II), and the control group (n = 12) received two daily saline injections from days 1 to 28. Lateral cephalometric, forelimb and hindlimb radiographs obtained weekly were scanned, standardized points digitized, and distances were measured using the Viewbox software. Growth curves between groups were compared using multilevel iterative generalized least squares curve fitting procedures. Supplementation during Phase I in the early group produced significant treatment effects in cranial and cranial base, midface, posterior corpus, and limb lengths which varied inversely with relative maturity (percentage growth completed at the start of the study). During Phase II, GH supplementation in the early and late groups showed treatment effects as above and additional viscerocranial and mandibular measurements, but these effects were unrelated to the relative maturity of the variables. These latter results are at variance with earlier findings in GH-deficient rats, raising the possibility that that GH-normal rats may not respond to GH supplementation in a similar fashion to GH-deficient rats.

Maturational and functional related differences in rat craniofacial growth.

BACKGROUND: Whilst decreases in masticatory muscle function have been linked with increased prevalences of craniofacial dysmorphology and malocclusion in humans, the relative susceptibility of the different craniofacial components remains poorly understood. METHODS: Thirty-two wild-type male Sprague-Dawley rats were randomly assigned to two groups (n=16 each), one raised on a soft diet (SD) and the other on a hard diet (HD). Body weights and three radiographs (lateral, dorso-ventral, and tibial X-rays) were taken at baseline (T1=23 days old) and every 2 weeks thereafter for 8 weeks (T5=79 days old). The X-ray images were scanned, standardised points were digitised, and linear measurements were calculated. Multilevel statistical models were used to describe longitudinal absolute growth changes and statistically evaluate group differences. Relative maturity curves were generated for each measurement based on the animals' T5 status. The experimental effect was calculated as the absolute and relative growth differences between the HD and SD groups. RESULTS: The HD group was significantly heavier than the SD group at T5, but no differences in tibial length were observed. Eight of the 20 craniofacial measurements (40%) showed significant size differences at the end of the experiment, with the SD group showing deficiencies in each instance. All of the vertical measurements, as well as most of the mandibular (67%) and transverse (67%) measures, showed absolute growth deficits in the SD group. Relative maturity curves demonstrated considerable variation among craniofacial structures (ranging from 42 to 98%). The neurocranial measures were the most mature; the mandibular measures were the least mature; the viscerocranial measures, which were most variable, tended to be intermediate. Whilst unrelated to the absolute experimental effect, the structures' relative maturity explained almost 70% (r=-0.82) of the relative experimental effect. CONCLUSION: The results of this study support the notion that masticatory function is a key determinant of the craniofacial growth pattern and that its effects are modulated by the relative growth potential of the different craniofacial components.

Craniofacial growth in growth hormone-deficient rats after growth hormone supplementation.

INTRODUCTION: Growth hormone (GH) supplementation is an established therapy to increase stature in GH-deficient or short-for-age children, but comparatively little is known of its effects on the craniofacial skeleton. METHODS: Using a mutant strain of Lewis rats (dw/dw) in which GH levels were 6% to 10% of normal, but other trophic hormones were unaffected, we investigated the differential susceptibility of craniofacial measures to GH supplementation, characterized their potential for partial or complete catch-up growth, and compared their growth changes with those in long bones. At 24 days of age and for 3 subsequent weeks, radiographs of the lateral head, upper limb, and lower limb were obtained from 3 groups of growing rats (n = 8-9 in each group): dwarf (experimental) with GH injection, dwarf (sham) with vehicle injection, and wild type (control) with vehicle injection. The x-ray images were scanned, standardized points digitized, and linear distances measured. Absolute growth curves were generated for each group by using multilevel modeling procedures and iterative generalized least-squares curve fitting. RESULTS: For every measure, growth differences were evident between the experimental and the sham groups, but the treatment effect varied inversely with relative maturity of the measure. Although all craniofacial measures showed some catch-up growth, only 31% of craniofacial measures had complete catch-up compared with all limb measures. The percentage of catch-up varied inversely with relative maturity of the measure. Our results suggest that the effects of GH supplementation vary considerably, so that measures with the lowest relative maturity (greatest baseline potential) show the greatest treatment effect and catch-up, whereas more mature measures show less growth response to GH replacement. CONCLUSIONS: These results suggest that, depending on the timing of GH supplementation, there is potential for change in proportions or shape of the craniofacial complex.