Earl Freymiller

Differential Expression of Fibromodulin, a Transforming Growth Factor-β Modulator, in Fetal Skin Development and Scarless Repair

Transforming growth factor-beta (TGF-beta1, -beta2, and -beta3) has been implicated in the ontogenetic transition from scarless fetal repair to adult repair with scar. Generally, TGF-beta exerts its effects through type I and II receptors; however, TGF-beta modulators such as latent TGF-beta binding protein-1 (LTBP-1), decorin, biglycan, and fibromodulin can bind and potentially inhibit TGF-beta activity. To more fully explore the role of TGF-beta ligands, receptors, and potential modulators during skin development and wound healing, we have used a rat model that transitions from scarless fetal-type repair to adult-type repair with scar between days 16 and 18 of gestation. We showed that TGF-beta ligand and receptor mRNA levels did not increase during the transition to adult-type repair in fetal skin, whereas LTBP-1 and fibromodulin expression decreased. In addition, TGF-beta1 and -beta3; type I, II, and III receptors; as well as LTBP-1, decorin, and biglycan were up-regulated during adult wound healing. In marked contrast, fibromodulin expression was initially down-regulated in adult repair. Immunostaining demonstrated significant fibromodulin induction 36 hours after injury in gestation day 16, but not day 19, fetal wounds. This inverse relationship between fibromodulin expression and scarring in both fetal and adult rat wound repair suggests that fibromodulin may be a biologically relevant modulator of TGF-beta activity during scar formation.

Ontogenetic Transition in Fetal Wound Transforming Growth Factor-β Regulation Correlates with Collagen Organization

Fetal rat skin transitions from scarless fetal-type repair to adult-type repair with scar between day 16 (E16) and day 18 (E18) of gestation (term = 21.5 days). Deficient transforming growth factor (TGF)-beta 1 and -beta 2 injury response has been proposed as a mechanism for scarless fetal-type repair. However, previous fetal studies have inconsistently reported the degree of TGF-beta induction after injury. To minimize developmental variables in fetal versus adult TGF-beta regulation, we narrowed our study to wounded fetal animals. We hypothesize that TGF-beta ligand and receptor expression will be differentially regulated during the transition from early gestation (E16) wounds manifesting scarless fetal-type repair to late gestation (E19) wounds manifesting adult-type repair with scar. In this study, decreased and rapidly cleared TGF-beta 1 and -beta 2 expression accompanied by increased and prolonged TGF-beta 3 levels in wounded E16 animals correlated with organized collagen deposition. In contrast, increased and prolonged TGF-beta 1 and -beta 2 expression accompanied by decreased and delayed TGF-beta 3 expression in wounded E19 animals correlated with disorganized collagen architecture. Similarly, expression of TGF-beta receptors type I and II were also increased or prolonged in E19 animals. Our results implicate increased TGF-beta 1, -beta 2, and decreased TGF-beta 3 expression, as well as increased type I and II receptor expression in late gestation fetal scar formation.

Cutaneous rat wounds express C49a, a novel gene with homology to the human melanoma differentiation associated gene, Mda‐7

We have used DD‐PCR (differential display‐polymerase chain reaction) to identify new genes that are over‐ or underexpressed during wound repair. DD‐PCR performed on excisional wounds identified the expression of rat c49a. Cloning and sequence analysis of the rat c49a gene revealed high homology to a novel human melanoma differentiation associated gene, mda‐7. The human mda‐7 gene isolated from melanoma cell lines, has been linked with human melanoma differentiation, and growth suppression. Moreover, transfection of human mda‐7 constructs into human tumor cells suppresses the growth and colony formation of tumor cells from diverse origins. To confirm and relatively quantitate expression of rat c49a gene during repair, specific primer, reduced cycle RT‐PCR (reverse transcription‐PCR) was performed. RT‐PCR showed an ∼9 to 12‐fold elevation of rat c49a mRNA at 12 h to 5 days above nonwounded controls that gradually decreased to ∼1.5 to 3‐fold by day 14. Cloning and sequence analysis of the entire 1200 base pair c49a gene product showed 78% nucleotide homology to human mda‐7. Immunohistochemistry studies localized rat C49A expression primarily to fibroblast‐like cells at the wound edge and base. The marked up‐regulation of rat c49a transcripts during the inflammatory and early granulation tissue phases of wound repair where cellular processes such as re‐epithelialization, angiogenesis, and fibroplasia predominate—suggest that c49a is associated with proliferation of fibroblasts in wound healing. J. Cell. Biochem. 74:1–10, 1999.

A randomized clinical trial comparing the efficacy of mandibular implant-supported overdentures and conventional dentures in diabetic patients. Part I: Methodology and clinical outcomes.

STATEMENT OF PROBLEM Scientific evidence is lacking to support the general application of implant-supported mandibular overdentures. PURPOSE This randomized clinical trial was undertaken to compare the efficacy of conventional mandibular and implant-supported overdentures in diabetic edentulous patients with clinically acceptable metabolic control. METHOD A total of 102 diabetic patients, treated with or without insulin, were randomized to receive a new maxillary denture and either a conventional or an implant-supported removable mandibular overdenture. Treatment was completed for 89 patients, 37 with the conventional and 52 with implant-supported dentures. Detailed examinations, tests, and questionnaires were given before and at 6- and 24-months after treatment completion. Comparisons between the two treatment groups were made for treatment failures based on prespecifed criteria and the type and amount of maintenance care provided. RESULTS The insulin and noninsulin treated groups were collapsed because of the lack of significant differences at entry. The conventional denture and implant-supported overdenture groups were similar in terms of general demographics, medical status, quality of their original dentures and denture support, several functional measures, and patient satisfaction. Treatment was judged to be successful in 56.9% of patients with conventional dentures and 72.1% with overdentures. This difference in success rate was not statistically significant (p > 0.05). Patients with treatment failures in both groups required excessive maintenance care. Those with conventional dentures needed frequent denture base adjustments and relines, whereas those with overdentures required frequent clip replacements and repairs. Although significant improvements were seen with both treatment modalities, a higher percentage of patients with implant-supported overdentures than those with conventional dentures reported improvements in chewing comfort and moderate-to-complete overall satisfaction.

Randomized clinical trial comparing the efficacy of mandibular implant-supported overdentures and conventional dentures in diabetic patients. Part III: Comparisons of patient satisfaction

STATEMENT OF PROBLEM There is insufficient evidence to indicate the functional superiority of mandibular implant-supported overdentures to justify their use in edentulous patients. PURPOSE This study compared the benefits perceived by patients who received a new maxillary denture and a mandibular conventional denture (CD) and an implant-supported overdenture (IOD). METHOD New maxillary and mandibular dentures were delivered to 89 diabetic denture wearers with clinically acceptable metabolic control who treated their diabetes either with insulin (IT) or without insulin (NIT). Of the 89 patients, 37 received maxillary and mandibular CDs and 52 received a maxillary CD and an IOD. Two questionnaires with categorical responses were used; the first contained 13 questions to ascertain a patients absolute assessments of original dentures at entry and study dentures at 6- and 24-months after treatment completion; the second questionnaire had 11 questions that assessed the relative change perceived by patients with study dentures. Of the 78 patients who completed the posttreatment (PT) assessments at 6 months, 68 patients provided longitudinal data for questionnaire I and cross-sectional data for questionnaire II. In addition, 46 patients (18 CD and 28 IOD) also provided PT assessments at 24 months. RESULTS Both mean scores and percentage distributions of longitudinal data for questionnaire I showed perceptual improvements with both types of study dentures. Improvements were higher in the IOD than in the CD group. Mean scores failed to show any significant differences between the 2 treatment groups. The only significant difference was found in the change in percentage distributions for perceptual chewing ability in favor of the IOD group. Even this advantage was lost at 24 months. With the comparative questionnaire, a higher percentage of patients in the IOD group than in the CD group perceived improvements with study dentures from their original dentures in chewing ability, chewing comfort, and denture security. However, mean differences were statistically significant in favor of the IOD group only for chewing ability and less difficulty to chew hard foods. CONCLUSION The mandibular implant-supported overdenture offers same advantage in terms of perceived chewing function over the conventional denture.

Teeth in the line of fracture: A review

The management of teeth in the line of fracture is still controversial. This lack of consensus about what is appropriate therapy led to a review of the literature. The various opinions expressed are reappraised in the context of the new treatment modalities available today, and recommendations are made to help the surgeon in his decision making.

Osseointegrated implants and orbital defects: U.C.L.A. experience

PURPOSE A clinical study of 23 craniofacial implants placed in 8 irradiated and nonirradiated orbital detects was conducted over a 7-year period. MATERIAL AND METHODS Implant-retained orbital prostheses were fabricated, implant success rate was determined, and the soft tissue responses were recorded at 6-month intervals. As a result of patient death, no data were gathered on three implants. A five-point scale was used to record the health of the peri-implant soft tissues and the patients were followed from 9 to 72 months. The unit of measure was a visit/site that was assigned for each instance an implant site was evaluated. Evaluations were conducted at 6-month intervals, and for the study period, there were 80 visit/sites. RESULTS The study revealed that 42.5% (34/80) of the visit/sites demonstrated an absence of inflammation; 23.7% (19/80) of visit/sites demonstrated slight redness; 13.8% (11/80) demonstrated peri-implant red and moist tissues; 6.2% (5/80) demonstrated granulation tissue associated with the implants; and 13.8% (11/80) infection of the peri-implant soft tissues was noted. Implant success rate was 35% (7/20); implant success rate in the nonradiated patients was 37.5% (3/8) and the success rate for radiated patients was 33.3% (4/12). Implants placed in the orbital region demonstrated a high failure rate. Most implant failures occurred late as opposed to early in the study period. CONCLUSION Orbital implants should be placed in patients who understand that long-term success rates may be low and require meticulous hygiene maintenance.

The effect of NELL1 and bone morphogenetic protein-2 on calvarial bone regeneration.

PURPOSE Most craniofacial birth defects contain skeletal components that require bone grafting. Although many growth factors have shown potential for use in bone regeneration, bone morphogenetic proteins (BMPs) are the most osteoinductive. However, supraphysiologic doses, high cost, and potential adverse effects stimulate clinicians and researchers to identify complementary molecules that allow a reduction in dose of BMP-2. Because NELL1 plays a key role as a regulator of craniofacial skeletal morphogenesis, especially in committed chondrogenic and osteogenic differentiation, and a previous synergistic mechanism has been identified, NELL1 is an ideal molecule for combination with BMP-2 in calvarial defect regeneration. We investigated the effect of NELL1 and BMP-2 on bone regeneration in vivo. MATERIALS AND METHODS BMP-2 doses of 589 and 1,178 ng were grafted into 5-mm critical-sized rat calvarial defects, as compared with 589 ng of NELL1 plus 589 ng of BMP-2 and 1,178 ng of NELL1 plus 1,178 ng of BMP-2, and bone regeneration was analyzed. RESULTS Live micro-computed tomography data showed increased bone formation throughout 4 to 8 weeks in all groups but a significant improvement when the lower doses of each molecule were combined. High-resolution micro-computed tomography and histology showed more mature and complete defect healing when the combination of NELL1 plus BMP-2 was compared with BMP-2 alone at lower doses. CONCLUSION The observed potential synergy has significant value in the future treatment of patients with craniofacial defects requiring extensive bone grafting that would normally entail extraoral autogenous bone grafts or doses of BMP-2 in milligrams.

A randomized clinical trial comparing the efficacy of mandibular implant-supported overdentures and conventional dentures in diabetic patients. Part II. Comparisons of masticatory performance ☆1 ☆2 ☆3 ☆4 ☆5 ☆6 ☆7 ☆8 ☆9 ☆10 ☆11 ☆12

Statement of problem. It is unclear whether the replacement of conventional mandibular dentures with implant-supported overdentures alters the diet and thus improves the nutritional intake of edentulous persons. Purpose. The purpose of this study was to compare the pretreatment and posttreatment diets of edentulous diabetic patients who received new dentures with either a conventional complete mandibular denture (CD) or a mandibular implant-supported overdenture (IOD). Material and methods. New dentures were made for 89 edentulous diabetic patients with acceptable metabolic control without insulin (NIT) or with insulin (IT). A randomized approach was used to assign 37 patients a mandibular CD and 52 patients a mandibular IOD supported by 2 cylindrical implants. Of the 89 patients, 58 submitted a dietary log for 7 consecutive days before treatment (PT) and 6 months after treatment completion (PTC). An average daily intake of 28 essential nutrients was determined for each patient at each time interval. Separate 2 × 2 × 2 repeated analysis of variance (ANOVA) tests were performed for each nutrient to compare the means of the 2 denture groups (CD and IOD), 2 diabetic groups (NIT and IT), and 2 time intervals (PT and PTC). The intakes were also compared with the recommended daily allowance (RDA). Results. ANOVAs for all 28 nutritional variables showed no main effect for either denture type or diabetic treatment. Time effects were seen for magnesium, potassium, copper, and monounsaturated fats. The PTC mean intake of the total sample (N = 58) decreased for all 3 minerals and increased for monounsaturated fats with study dentures. Post hoc tests showed the differences between PT and PTC means to be statistically significant for only magnesium (P=.043) and potassium (P=.015). The percentage of patients with PT intake 25% or more below the RDA ranged from 33% to 85% in the CD group and from 24% to 100% in the IOD group for the same 11 nutrients. PTC fiber intake deficiency was noted in almost all participants. Carbohydrate consumption was markedly lower than that recommended by the American Diabetic Association. Conclusion. As is often the case with elderly groups, this group of edentulous diabetic patients showed highly comprised nutritional intakes of fiber, vitamins, and minerals. The replacement of old dentures with new dentures that included either a mandibular CD or IOD did not alter patient diets such that the patients improved their nutritional intakes of essential micronutrients and macronutrients. (J Prosthet Dent 2001;85:53-60.)

The identification of novel wound-healing genes through differential display.

Effective methods to identify novel genes in complicated dynamic tissue processes are needed in molecular biology research. Traditional techniques primarily target known genes and are inefficient in the pursuit of unknown genes. Here we describe the use of a modified differential display polymerase chain reaction (DD-PCR) protocol for the identification of genes differentially expressed in wound healing. Full-thickness dorsal wounds were made on 35 adult rats, followed by wound harvest at 12 hours, 24 hours, 3 days, 5 days, 7 days, 10 days, and 14 days after injury. Modified DD-PCR was performed and gene fragments displaying definite changes during wound healing were cloned and sequenced. Gene fragments from DD-PCR were compared with available gene bank database sequences. Specific primer PCR was used to confirm DD-PCR expression patterns. As a result, over 1000 gene fragments were amplified by DD-PCR, 35 of which demonstrated distinct differences during repair. Cloning and sequencing of 13 of these gene fragments revealed that some were homologous to several characterized genes with previously unsuspected roles in repair, whereas others were completely novel genes with no known function. Specific primer PCR further confirmed expression of six of these 13 gene fragments. Only one of the 13 cloned fragments, later identified as interleukin-1beta, had well-recognized associations with tissue injury. Other fragments corresponded to various genes involved in cellular processes such as differentiation, proliferation, exocytosis, and myofibril assembly. No prior studies have linked them to wound healing. We have demonstrated that modified DD-PCR can be used to effectively identify novel genes differentially expressed during repair. Because DD-PCR allows for the simultaneous amplification of multiple arbitrary transcripts, it is a powerful genetic screening tool for complicated dynamic tissue processes, particularly when multiple, limited-sized samples are involved.