A Prospective, Open-Label Study to Evaluate the Impact of VYC-12L Injection on Skin Quality Attributes in Healthy Volunteers.

Purpose: Age-related changes in skin structure and function can negatively impact skin quality. VYC-12L is a crosslinked hyaluronic acid filler for treating fine lines and improving hydration and elasticity. The goal of this study was to understand skin quality, histologic, and genomic changes underlying long-term clinical benefits of VYC-12L treatment. Patients and Methods: In this prospective, nonrandomized, open-label study, 11 healthy men (n = 2) and women (n = 9) received intradermal VYC-12L treatment on the volar forearm. Clinical probes assessed skin quality at baseline and months 1 and 3 post-treatment. Punch biopsies were collected 1 and 3 months post-treatment to evaluate histologic and genomic changes. Safety was evaluated throughout. Results: Participants had a mean age of 41 years and Fitzpatrick skin phototypes II (54.5%) and III (45.5%). At months 1 and 3, VYC-12L-treated skin had higher hydration in the stratum corneum than untreated skin. Cutometer measurements indicated treated skin that was firmer and more resistant to deformation. Histology showed increased epidermal AQP3 and Ki67 expression 1 and 3 months post-treatment and a qualitative increase in papillary dermal collagen I at month 3. Genomic analyses demonstrated treatment-related upregulation of genes involved in adipocyte differentiation, lipid metabolism, keratinocyte renewal, and dermal extracellular matrix (ECM) maintenance. Injection site reactions were mild-to-moderate in severity and resolved by month 1. Five participants reported 19 adverse events; most (68.4%) were related to the biopsy and none to VYC-12L. Conclusion: VYC-12L produced changes in hydration, firmness, and ECM density and composition consistent with improved skin properties, demonstrating that VYC-12L can act as a substrate for tissue repair.

Novel Recombinant Tropoelastin Implants Restore Skin Extracellular Matrix.

BACKGROUND: Elastin is an essential component of the dermis, providing skin with elasticity and integrity. Elastin and other dermal components are gradually lost through aging, sun damage, and following injury, highlighting a need to replace these components to repair the skin. Tropoelastin (TE) in monomeric form was previously shown to be utilized as a substrate by dermal fibroblasts during the production of elastin fibers in vitro. OBJECTIVE: To analyze coaccumulation of elastin and collagen and gene expression of biomarkers associated with elastin production, examine the ex vivo effects of recombinant human TE (rhTE) and hyaluronic acid (HA) on epidermal and dermal structures, and evaluate the in vivo response following intradermal injections of rhTE and HA. METHODS: Human dermal fibroblasts and 3-D skin patch models were cultured for in vitro analysis. Ex vivo analysis was performed using skin explants. In vivo studies were done in 6-week-old male CD Hairless rats. Different formulations of rhTE, soluble or crosslinked using derivatized HA (dHA), were tested and analyzed. RESULTS: rhTE in monomeric form was utilized as a substrate by dermal fibroblasts during the production of branched elastin and fibrous collagen networks in vitro. Formulations of rhTE crosslinked with dHA demonstrated increased expression of hyaluronic acid synthase 1 and ex vivo results revealed increased moisture content and glycosaminoglycan (GAG) deposition versus dermal filler control. Intradermal rhTE‒dHA injection produced colocalized human‒rat elastin fibers in vivo. CONCLUSIONS: These results suggest that the novel rhTE‒dHA matrix is an attractive material to support skin tissue repair.J Drugs Dermatol. 2020;19(12): doi:10.36849/JDD.2020.5375.

Evaluation of Physicochemical Properties Following Syringe-to-Syringe Mixing of Hyaluronic Acid Dermal Fillers.

BACKGROUND: Historically, soft-tissue hyaluronic acid (HA) fillers have been mixed with agents to reduce pain or alter physicochemical properties. OBJECTIVE: Evaluate the impact of dilution and mixing on HA filler physicochemical properties. MATERIALS AND METHODS: Crosslinked HA filler (VYC-20L, 20 mg/mL) was diluted to 15 mg/mL using saline through 5 or 10 passes between 2 syringes connected using a luer connector. Extrusion force, rheological properties, and microscopic appearance were assessed. Undiluted VYC-15L (15 mg/mL) served as the control. RESULTS: Average extrusion force was higher for diluted VYC-20L versus the control, with an increase in slope for gel diluted using 5 passes (0.65) and 10 passes (0.52) versus the control (<0.1). For diluted samples mixed with 5 or 10 passes, the rheological profile was different between the 2 halves of the syringe, with the second half more elastic than the first half, compared with the consistent profile of undiluted samples. Microscopically, diluted VYC-20L samples seemed more liquid near the luer and more particulate near the piston compared with the control, which was smooth throughout. CONCLUSION: In addition to potentially introducing contamination, diluting or mixing soft-tissue HA fillers yields a heterogeneous product with physicochemical characteristics that vary substantially throughout the syringe.

Improvements in Skin Quality Biological Markers in Skin Explants Using Hyaluronic Acid Filler VYC-12L.

Hyaluronic acid (HA), both crosslinked and uncrosslinked, is used clinically to treat fine lines and provides additional improvements in skin quality attributes. The purpose of this study was to assess potential early differences in the expression of biological markers of skin quality in living human skin explants injected with uncrosslinked and crosslinked HA gels. METHODS: Living human skin explants injected with VYC-12L or noncrosslinked HA with mannitol (HYD) and noninjected controls were assessed via microscopy, histology, and immunohistochemistry on days 3 and/or 8 for biological markers of elasticity (collagen density, elastin, fibrillin-1) and hydration [aquaporin-3, acidic glycosaminoglycans (GAGs), HA]. Hydration was also assessed via a corneometer probe on days 0, 1, 2, and 8. RESULTS: On day 3 versus controls, VYC-12L moderately increased collagen density in the upper reticular dermis and clearly increased fibrillin-1 expression, with slight increases persisting on day 8. Increases with HYD were smaller and did not persist on day 8. Both VYC-12L and HYD increased aquaporin-3 expression and GAG content on days 3 and 8, but VYC-12L produced greater GAG increases in the reticular dermis. Day 8 instrument-assessed hydration increased by 49% and 22% for VYC-12L and HYD, respectively. Elastin expression in oxytalan and elaunin fibers was unchanged. Upper-dermal HA reductions suggested HA injection-induced hyaluronidase expression. CONCLUSION: VYC-12L produced greater, more lasting improvements in biological markers of skin quality than HYD.

In Vivo Degradation of Crosslinked Hyaluronic Acid Fillers by Exogenous Hyaluronidases.

BACKGROUND: An advantage of hyaluronic acid (HA)-based fillers is reversibility. OBJECTIVE: To evaluate the ability of 2 hyaluronidases to degrade 3 HA-based fillers using a novel in vivo model. MATERIALS AND METHODS: Rats were injected with 3 HA fillers (HYC-24L+, VYC-20L, and RES-L) to create a projecting bolus. After 4 days, recombinant human hyaluronidase (HX) or ovine hyaluronidase (VIT) was administered at (1) varying doses (5 U, 10 U, or 30 U per 0.1 mL filler) or (2) different dilutions (10 U diluted 3-fold). The impact of tissue integration was assessed by administering 10 U/0.1 mL filler 4 weeks after filler injection. Three-dimensional images quantified projection loss over 72 hours. RESULTS: Complete loss of projection was achieved for all fillers with the highest HX and VIT doses; lower doses achieved less degradation. No difference in degradation was observed between HYC-24L+ and VYC-20L using HX or VIT. RES-L was slightly more degraded with 10 U VIT but not with 10 U HX. Enzyme dilution resulted in less degradation. Tissue integration did not impact the degree of degradation. CONCLUSION: This model incorporates the biological system while controlling variables including filler depth and volume and location of hyaluronidase delivery. Hyaluronic acid filler degradation by exogenous hyaluronidase was not hindered by differences among fillers.

Rheological Properties and In Vivo Performance Characteristics of Soft Tissue Fillers.

BACKGROUND: Physicochemical properties and performance in nonclinical animal models can provide insights into soft tissue filler performance. OBJECTIVE: To evaluate the in vivo performance of fillers with different compositions and physicochemical properties. MATERIALS AND METHODS: Physicochemical properties were measured in vitro. Rat models were developed and used to compare lift capacity, resistance to deformation, and tissue integration. Four homogeneous hyaluronic acid (HA) fillers, 2 nonanimal stabilized HA (NASHA) fillers, and 1 calcium hydroxylapatite/carboxymethyl cellulose (CaHA/CMC) filler were evaluated. RESULTS: Filler lift capacity correlated better with filler composition/type (homogeneous > NASHA > CaHA/CMC) than with specific rheological properties. The CaHA/CMC filler had high initial resistance to deformation relative to other groups; all HA fillers exhibited lower initial resistance to deformation, which increased over time. Homogeneous HA fillers were integrated with surrounding tissue, whereas integration within particle-based fillers (NASHA and CaHA/CMC) was variable, with some areas void of tissue. CONCLUSION: The animal models provide a platform to make comparative evaluations among fillers. The results indicated that biological interaction plays an important role in how the filler performs. Rheology alone was not sufficient to understand filler performance but was most useful when comparing within fillers of similar composition.

Skin extracellular matrix stimulation following injection of a hyaluronic acid-based dermal filler in a rat model.

BACKGROUND: Hyaluronic acid-based dermal fillers have gained rapid acceptance for treating facial wrinkles and deep tissue folds. Although their space-filling properties are well understood, this study evaluates the cellular and molecular changes in skin, as a secondary effect, following injection of a commercially available, 24-mg/ml, cross-linked hyaluronic acid-based filler (HYC-24L+) in a rodent model. METHODS: Sprague-Dawley rats, aged 2 to 4 months, were injected intradermally with 20 µl of HYC-24L+ using a linear threading technique and followed to 12 weeks after injection. Untreated skin and saline injection were used as study controls. Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction methods were used to investigate changes in the expression of several extracellular matrix proteins and genes over time. RESULTS: HYC-24L+ significantly increased the protein expression levels of collagen types I and III in rat dermal tissue for up to 12 weeks. The ratio of collagen type III to type I protein, however, remained unchanged, suggesting maintenance of collagen homeostasis. A significant increase in dermal elastin after HYC-24L+ injection was also observed. Gene expression analysis confirmed that several genes associated with extracellular matrix production and assembly were also transiently up-regulated, and that these changes temporally preceded those observed at the protein level. CONCLUSION: In addition to its well-understood space-filling function, as a secondary effect, the authors demonstrate that HYC-24L+ stimulates the production of several extracellular matrix components, including dermal collagen and elastin.

Comparison of the effect of intra-tendon applications of recombinant human platelet-derived growth factor-BB, platelet-rich plasma, steroids in a rat achilles tendon collagenase model.

This study compared the effect of intra-tendon (IT) delivery of recombinant human platelet-derived growth factor-BB (rhPDGF-BB), platelet-rich plasma (PRP) and corticosteroids in a rat tendinopathy model. Seven days after collagenase induction of tendinopathy, a 30-µl IT injection was administered. Treatments included: saline; 3 µg rhPDGF-BB; 10 µg rhPDGF-BB; PRP; and 300 µg triamcinolone acetonide (TCA). Outcomes were assessed 7 and 21 days after treatment. All groups exhibited good to excellent repair. Relative to saline, cell proliferation increased 65% in the 10 µg rhPDGF-BB group and decreased 74% in the TCA group; inflammation decreased 65% in the TCA group. At 7 days, maximum load-to-failure was increased in the 3 µg rhPDGF-BB group relative to saline, PRP, and TCA (p < 0.025). On day 21, maximum load-to-rupture was increased in the 10 µg rhPDGF-BB group relative to saline, PRP, and TCA (p < 0.035) and in the 3 µg rhPDGF-BB group compared to saline and TCA (p < 0.027). Stiffness in the 10 µg rhPDGF-BB group was increased compared to saline, PRP, and TCA (p < 0.038). Histology demonstrated similar repair in all groups. PRP and TCA did not improve mechanical properties compared to saline. Injections of rhPDGF-BB increased maximum load-to-failure (3 and 10 µg) and stiffness (10 µg) relative to controls and commonly used treatments. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:145-150, 2014.

Dose-response effect of an intra-tendon application of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) in a rat Achilles tendinopathy model.

The purpose of this study was to assess whether intra-tendon delivery of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) would improve Achilles tendon repair in a rat collagenase-induced tendinopathy model. Seven days following collagenase induction of tendinopathy, one of four intra-tendinous treatments was administered: (i) Vehicle control (sodium acetate buffer), (ii) 1.02 µg rhPDGF-BB, (iii) 10.2 µg rhPDGF-BB, or (iv) 102 µg rhPDGF-BB. Treated tendons were assessed for histopathological (e.g., proliferation, tendon thickness, collagen fiber density/orientation) and biomechanical (e.g., maximum load-to-failure and stiffness) outcomes. By 7 days post-treatment, there was a significant increase in cell proliferation with the 10.2 and 102 µg rhPDGF-BB-treated groups (p=0.049 and 0.015, respectively) and in thickness at the tendon midsubstance in the 10.2 µg of rhPDGF-BB group (p=0.005), compared to controls. All groups had equivalent outcomes by Day 21. There was a dose-dependent effect on the maximum load-to-failure, with no significant difference in the 1.02 and 102 µg rhPDGF-BB doses but the 10.2 µg rhPDGF-BB group had a significant increase in load-to-failure at 7 (p=0.003) and 21 days (p=0.019) compared to controls. The rhPDGF-BB treatment resulted in a dose-dependent, transient increase in cell proliferation and sustained improvement in biomechanical properties in a rat Achilles tendinopathy model, demonstrating the potential of rhPDGF-BB treatment in a tendinopathy application. Consequently, in this model, data suggest that rhPDGF-BB treatment is an effective therapy and thus, may be an option for clinical applications to treat tendinopathy.

Effect of recombinant human platelet-derived growth factor-BB-coated sutures on Achilles tendon healing in a rat model: A histological and biomechanical study.

PURPOSE: Repairing tendon injuries with recombinant human platelet-derived growth factor-BB has potential for improving surgical outcomes. Augmentation of sutures, a critical component of surgical tendon repair, by coating with growth factors may provide a clinically useful therapeutic device for improving tendon repair. Therefore, the purpose of this study was to (a) coat Vicryl sutures with a defined dose of recombinant human platelet-derived growth factor-BB without additional coating excipients (e.g. gelatin), (b) quantify the recombinant human platelet-derived growth factor-BB released from the suture, and (c) use the recombinant human platelet-derived growth factor-BB-coated sutures to enhance tendon repair in a rat Achilles tendon transection model. METHODS: Vicryl sutures were coated with 0, 0.3, 1.0, and 10.0 mg/mL concentrations of recombinant human platelet-derived growth factor-BB using a dip-coating process. In vitro release was quantified by an enzyme-linked immunosorbent assay. Acutely transected rat Achilles tendons were repaired using one of the four suture groups (n = 12 per group). Four weeks following repair, the tensile biomechanical and histological (i.e. collagen organization and angiogenesis) properties were determined. RESULTS: A dose-dependent bolus release of recombinant human platelet-derived growth factor-BB occurred within the first hour in vitro, followed by a gradual release over 48 h. There was a significant increase in ultimate tensile strength (p < 0.01) in the two highest recombinant human platelet-derived growth factor-BB dose groups (1.9 ± 0.5 and 2.1 ± 0.5 MPa) relative to controls (1.0 ± 0.2 MPa). The modulus significantly increased (p = 0.031) with the highest recombinant human platelet-derived growth factor-BB dose group (7.2 ± 3.8 MPa) relative to all other groups (control: 3.5 ± 0.9 MPa). No significant differences were identified for the maximum load or stiffness. The histological collagen and angiogenesis scores were comparable in all groups, although there was a trend for improved collagen organization in the recombinant human platelet-derived growth factor-BB-treated groups (p = 0.054). CONCLUSIONS: The results of this study suggest that recombinant human platelet-derived growth factor-BB can be used to reproducibly coat Vicryl sutures and improve remodeling in a rat Achilles tendon transection model by significantly decreasing the resulting cross-sectional area, thus improving the material properties of the repaired tendon.

Safety of recombinant human platelet-derived growth factor-BB in Augment(®) Bone Graft.

This article discusses nonclinical and clinical data regarding the safety of recombinant human platelet-derived growth factor-BB as a component of the Augment(®) Bone Graft (Augment). Augment is a bone graft substitute intended to be used as an alternative to autologous bone graft in the fusion of hindfoot and ankle joints. Nonclinical studies included assessment of the pharmacokinetic profile of intravenously administered recombinant human platelet-derived growth factor-BB in rat and dog, effects of intravenous administration of recombinant human platelet-derived growth factor-BB in a reproductive and development toxicity study in rats, and chronic toxicity and carcinogenicity of Augment in a 12-month implantation model. These studies showed that systemic exposure was brief and clearance was rapid. No signs of toxicity, carcinogenicity, or tumor promotion were observed even with doses far exceeding the maximum clinical dose. Results of clinical trials (605 participants) and commercial use of recombinant human platelet-derived growth factor-BB containing products indicate that these products are not associated with increased incidence of adverse events or cancer. The safety data presented provide evidence that recombinant human platelet-derived growth factor-BB is a safe therapeutic when used in combination products as a single administration during surgical procedures for bone repair and fusion. There is no evidence associating use of recombinant human platelet-derived growth factor-BB in Augment with chronic toxicity, carcinogenicity, or tumor promotion.

Growth factors and craniofacial surgery.

The specialty of craniofacial surgery is broad and includes trauma, aesthetics, reconstruction of congenital deformities, and regeneration of tissues. Moreover, craniofacial surgery deals with a diverse range of tissues including both "soft" and "hard" tissues. Technological advances in materials and biological sciences and improved surgical techniques have remarkably improved clinical outcomes. The quest to raise the bar for patient care continues to inspire advances for predictable biological regeneration of soft and hard tissues. As a consequence of this quest for advancement, a wide spectrum of biologicals is becoming available to surgeons. Is the use of recombinant DNA engineered biologicals daring? Sensible? Logical? Timely? Safe? It is crucial for the practicing craniofacial surgeon to take a step back periodically and carefully review the biological factors that have the potential for dramatically altering the discipline of craniofacial surgery. With this emphasis, the coauthors of this article will focus on growth factor technology underscoring bone tissue regeneration. As the 21st-century matures, recombinant human biologicals will have an overwhelming impact on the practice of craniofacial surgery.

Augment bone graft products compare favorably with autologous bone graft in an ovine model of lumbar interbody spine fusion.

STUDY DESIGN: This study was designed to determine whether Augment Bone Graft (Augment, Biomimetic Therapeutics, Inc., Franklin, TN) and Augment Injectable Bone Graft (Augment Injectable, Biomimetic Therapeutics, Inc., Franklin, TN), 2 combination devices comprising recombinant human platelet-derived growth factor-BB and ß-tricalcium phosphate-containing matrices, promote bone bridging in an ovine model of lumbar spine fusion. Autologous bone graft (autograft) was used as a positive control. OBJECTIVE: The purpose of this study was to determine the ability of Augment products to promote fusion of the L2-L3 and L4-L5 vertebral bodies in an ovine model. SUMMARY OF BACKGROUND DATA: In interbody spine fusion, the intervertebral disc is removed and a spacer is inserted for support and to facilitate bone growth. The fusion is commonly enhanced with grafts. Autograft is the "gold standard" but it has limitations including availability and donor-site morbidity. Synthetic graft substitutes eliminate these complications. Augment products are combination devices including recombinant human platelet-derived growth factor-BB, a well-characterized chemotactic, mitogenic, and proangiogenic protein essential in wound and bone healing. METHODS: Twenty-two sheep received an uninstrumented, double-level, interbody lumbar spinal fusion procedure using a polyetheretherketone spacer, which was either empty or packed with iliac crest autograft, Augment or Augment Injectable. The same treatment was used at both levels. Animals were 24 weeks after surgery, and fusion was assessed by micro-computed tomography (micro-CT) and histology. RESULTS: Micro-CT and histologic assessment of fusion revealed that empty controls had significantly lower fusion rates. No differences were detected among autografts, Augment, and Augment Injectable-treated specimens. Residual ß-tricalcium phosphate particles embedded in the newly formed bone were visible in Augment- and Augment Injectable-treated specimens. CONCLUSION: Augment-treated specimens had the highest fusion scores. Treatment with either of the Augment products significantly promoted interbody spine fusion compared with empty spacers and was equivalent to autograft-induced fusion. No adverse events were noted.

Regenerative tendon and ligament healing: opportunities with recombinant human platelet-derived growth factor BB-homodimer.

Intrinsic tendon healing in response to injury is a reparative process that often results in formation of scar tissue with functional and mechanical properties inferior to those of the native tendon. Development of therapies that can promote regenerative, rather than reparative, healing hold the promise of improving patient recovery from tendon and ligament injuries by producing tissue that is morphologically and functionally equivalent to the native tissue. One therapeutic approach that has been a frequent topic of investigation in the preclinical literature is the use of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) to augment tendon and ligament repair. The chemotactic, mitogenic, and pro-angiogenic properties of rhPDGF-BB have been shown to result in recruitment and proliferation of tenogenic cells and a commensurate boost in extracellular matrix deposition and organization, improving the morphological and biomechanical properties of healing tendons and ligaments. The outcomes of the preclinical studies reviewed here strongly suggest that rhPDGF-BB will provide a new therapeutic opportunity to improve the treatment of injured tendons and ligaments.

Augmentation of a rotator cuff suture repair using rhPDGF-BB and a type I bovine collagen matrix in an ovine model.

BACKGROUND: Rotator cuff tears are a common source of shoulder pain. High rates (20%-94%) of structural failure of the repair have been attributed to multiple factors, including poor repair tissue quality and tendon-to-bone integration. Biologic augmentation using growth factors has potential to promote tendon-to-bone integration, improving the function and long-term success of the repair. One such growth factor is platelet-derived growth factor-BB (PDGF-BB), which has been shown to improve healing in tendon and bone repair models. HYPOTHESIS: Recombinant human PDGF-BB (rhPDGF-BB) combined with a highly porous type I bovine collagen matrix will improve the biomechanical function and morphologic appearance of the repair in a dose-dependent manner, relative to a suture-only control, after 12 weeks in an acute ovine model of rotator cuff repair. STUDY DESIGN: Controlled laboratory study. METHODS: An interpositional graft consisting of rhPDGF-BB and a type I collagen matrix was implanted in an ovine model of rotator cuff repair. Biomechanical and histologic analyses were performed to determine the functional and anatomic characteristics of the repair after 12 weeks. RESULTS: A significant increase in the ultimate load to failure was observed in repairs treated with 75 µg (1490.5 ± 224.5 N, P = .029) or 150 µg (1486.6 ± 229.0 N, P = .029) of rhPDGF-BB, relative to suture-only controls (910.4 ± 156.1 N) and the 500-µg rhPDGF-BB group (677.8 ± 105.9 N). The 75-µg and 150-µg rhPDGF-BB groups also exhibited increased tendon-to-bone interdigitation histologically. No differences in inflammation or cellularity were observed among treatments. CONCLUSION: This study demonstrated that an interpositional graft consisting of rhPDGF-BB (75 or 150 µg) and a type I collagen matrix was able to improve the biomechanical strength and anatomic appearance in an ovine model of rotator cuff repair compared to a suture-only control and the 500-µg rhPDGF-BB group. CLINICAL RELEVANCE: Recombinant human PDGF-BB combined with a type I collagen matrix has potential to be used to augment surgical repair of rotator cuff tears, thereby improving clinical success.

Differential surface antigen expression and 1α,25-dihydroxyvitamin D3 responsiveness distinguish human dermal fibroblasts with age-dependent osteogenic differentiation potential from marrow-derived stromal cells in vitro.

BACKGROUND AIMS: Recent studies have demonstrated that cells committed to a fibroblastic lineage, including dermal fibroblasts, may undergo osteoblastic differentiation when treated with steroid hormones. However, stem cells have also been isolated from the dermis, making it unclear whether osteoinduction of dermal fibroblasts is the result of transdifferentiation of committed fibroblasts or differentiation of resident multipotent stromal cells, which are morphologically indistinguishable. METHODS: Flow cytometry was used to characterize the expression of CD26, CD90 and CD105 on neonatal and adult human dermal fibroblasts and adult human bone marrow-derived stromal cells. These cells were then cultured with the steroid hormones 1α,25-dihydroxyvitamin D(3) and dexamethasone, and evaluated for protein expression and mineral deposition typical of an osteoblastic phenotype. RESULTS: The surface peptidase, dipeptidyl peptidase IV (CD26), was differentially expressed between human neonatal (98.22 ± 1.47%) and adult (90.73 ± 7.97%) dermal fibroblasts and adult bone marrow-derived stromal cells (6.84 ± 5.07%). In addition, neonatal dermal fibroblasts treated with vitamin D(3) expressed alkaline phosphatase, osteocalcin and bone sialoprotein, and deposited mineral, which is consistent with an osteoblastic phenotype. Such differentiation was not observed in adult dermal fibroblasts. In contrast, marrow-derived stromal cells required dexamethasone in order to undergo osteoblastic differentiation. CONCLUSIONS: Taken together, the differential surface antigen expression and disparate response to steroid hormones suggest that committed neonatal dermal fibroblasts are distinct from mesenchymal stromal cells and possess osteogenic differentiation potential.

A rat model of temporomandibular joint pain with histopathologic modifications.

AIMS: To develop a rat model of temporomandibular joint (TMJ) pain and to characterize in it the development and temporal response of behavioral hypersensitivity as well as to evaluate if and to what extent a loading protocol is associated with histological changes in the TMJ consistent with osteoarthritic pathology. METHODS: A novel rat model of TMJ pain was developed using a noninvasive, mechanical loading protocol. Rats were exposed to steady mouth-opening for 7 days (2 N force, 1 hour/day), and mechanical hyperalgesia (increased pain response) was measured during the loading period and for 14 days thereafter. Histological modifications in the joint cartilage were also evaluated. Outcomes for the mouth-opening exposure were compared to age-matched controls. Thresholds for evoking responses were compared using a ranked ANOVA with repeated measures. RESULTS: Increased mechanical hypersensitivity in the temporomandibular region developed during daily loading and persisted even after the termination of the loading protocol. Histologic characterization revealed thinning of the cartilaginous structures of the joint and irregular zonal cellular arrangements in the condylar cartilage of rats subjected to the daily loading protocol. CONCLUSION: The injury model presented here is the first to demonstrate mechanically-induced behavioral hypersensitivity accompanied by osteoarthritic pathology in the TMJ.

Endogenous bone morphogenetic proteins mediate 1alpha, 25-dihydroxyvitamin D(3)-induced expression of osteoblast differentiation markers in human dermal fibroblasts.

Human dermal fibroblasts are generally considered to be restricted to a fibroblastic lineage. Although dermal fibroblasts do not typically express markers of osteoblastic differentiation, they have previously been shown to undergo osteoinduction when stimulated with bone morphogenetic proteins (BMPs) or vitamin D(3). However, involvement of BMP signaling in vitamin D(3)-mediated osteoinduction has not been reported. In this study, human dermal fibroblasts were cultured in chemically defined medium containing vitamin D(3), in the presence of the BMP antagonist noggin or neutralizing antibodies specific for BMP-4 or BMP-6, and characterized for markers of osteoblastic differentiation. Treatment of dermal fibroblasts with vitamin D(3) induced expression of BMP-4 (1.2 +/- 0.2, 1.7 +/- 0.2, and 1.8 +/- 0.2 relative fold increase) and BMP-6 (9.1 +/- 0.3, 23.3 +/- 2.1, and 30.4 +/- 3.0 relative fold increase) at 3, 14, and 21 days, respectively. Vitamin D(3) was also shown to induce the expression of the osteoblast-specific markers, alkaline phosphatase and osteocalcin, in a dose-dependent manner in human dermal fibroblasts. Addition of noggin, BMP-4 antibodies, and BMP-6 antibodies resulted in a downregulation of alkaline phosphatase activity (by 42%, 22%, and 20%, respectively) and secreted osteocalcin (by 20%, 31%, and 49%, respectively) after 21 days in culture. However, blocking BMP signaling did not result in complete recovery of a fibroblastic phenotype. Taken together, these results suggest that BMP signaling plays a role in the induction of an osteoblastic phenotype in human dermal fibroblasts in response to vitamin D(3) stimulation.

Influence of three-dimensional scaffold on the expression of osteogenic differentiation markers by human dermal fibroblasts.

Current research in the field of tissue engineering utilizes biomaterial scaffolds, cells, and growth factors for the creation of a functional, biologically active tissue. This study examined the effect of two commercially available, three-dimensional scaffolds, ultraporous beta-tricalcium phosphate ceramics (beta-TCP, Vitoss) and open-celled poly(lactic acid) foams (OPLA, Drilac), on the osteogenic differentiation potential of human dermal fibroblasts. Serum-free, chemically-defined medium containing the metabolic factor 1alpha,25-dihydroxyvitamin D3 was used to promote an osteogenic phenotype in these cells. Osteoblast differentiation was assessed using PCR and immunohistochemical methods to detect gene and protein expression for the osteoblast markers alkaline phosphatase, osteopontin, and osteocalcin. Dermal fibroblasts cultured on beta-TCP scaffolds in chemically-defined medium with vitamin D3 exhibited up-regulated gene and protein expression compared to cells cultured on OPLA scaffolds. These results suggest that Vitoss (beta-TCP) scaffolds seeded with dermal fibroblasts and maintained in chemically-defined medium with vitamin D3 are better suited for bone tissue engineering applications than Drilac (OPLA) foams.

Induction of osteoblast differentiation markers in human dermal fibroblasts: potential application to bone tissue engineering.

Tissue engineered constructs have the potential to be used as replacements for current bone graft technologies. One component necessary for bone tissue engineering is a readily available, osteogenic cell source. Human dermal fibroblasts may have the potential to differentiate along an osteoblastic lineage, making them a candidate for use in bone tissue engineering applications. The objective of this study was to validate the ability of dermal fibroblasts to express gene and protein markers of osteoblastic differentiation and to explore their potential, in combination with biomaterial scaffolds and signaling factors, for use in bone tissue engineering.