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Importance: In previous studies, this group has reported that cultured human fibroblasts suspended in hyaluronic acid (HA) filler might have potential use as a long-lasting injectable soft-tissue filler. However, the data were insufficient to determine the long-term outcomes., Objective: To evaluate the long-term outcomes of cultured human fibroblasts suspended in HA filler used for soft-tissue augmentation., Design, Setting, and Participants: A long-term case series study was performed. Between January 2010 and December 2013, a total of 38 patients were treated with fibroblast-HA filler mixture to augment nasal dorsa, nasolabial folds, and cheek depressions. Of these 38 patients, patients with follow-up period of greater than 3 years were included in this study. A total of 22 patients met the inclusion criteria., Main Outcomes and Measures: Subjective assessment was performed to evaluate degree and time of resorption, improvement, satisfaction, softness of injection sites, and willingness to recommend this treatment to others. Objective assessment was carried out with patients' photographs. Safety and tolerability were also evaluated for this treatment., Results: Of the 22 patients included in this study, 19 were women; mean (SD) patient age was 43 (15) years. All 22 patients experienced improvement following the treatment. Twenty (91%) patients were satisfied with the treatment. Nineteen patients (86%) considered that the injection site was as soft as it was before treatment. Patients' mean (SD) grading of improvement, satisfaction, and softness were 4.50 (0.51) (95% CI, 4.27-4.73), 4.14 (0.71) (95% CI, 3.82-4.45), and 4.82 (0.50) (95% CI, 4.59-5.00) at the last visit, respectively. Objective assessment demonstrated postoperative improvement in all patients: a rating of "much improved" was given to 7 patients (32%) by investigator 1; 8 patients (36%) by investigator 2; and 12 patients (55%) the injecting physician. This treatment was well tolerated; no adverse event was recorded for any patient., Conclusions and Relevance: Injection of cultured human fibroblasts suspended in HA filler might be successful for long-term soft-tissue augmentation. To our knowledge, this study represents the longest follow-up study of soft-tissue augmentation with a fibroblast-HA filler mixture to date., Level of Evidence: 4.

Background: A new commercial drug that contains autologous cultured fibroblasts has been developed and approved by the United States Food and Drug Administration for improving the appearance of nasolabial folds. However, the treatment requires three sessions every 3-6 weeks. It is known that the skin overlying the nasojugal groove is thinner, and the wrinkle is generally shallower than nasolabial folds. Therefore, we hypothesized that the nasojugal groove could be improved by just one treatment session. Therefore, the purpose of this study was to evaluate the efficacy and safety of autologous cultured fibroblast injection to correct nasojugal grooves., Methods: Forty-six subjects with nasojugal grooves were enrolled in this study. They were injected with autologous cultured fibroblasts or placebo in one session. Blinded evaluators and subjects assessed the efficacy using a validated wrinkle assessment scale at 4, 12, and 24 weeks after the injection. Information of adverse events was collected at each visit., Results: Based on the evaluators' assessment at 24 weeks after the injection, 76% of subjects treated with autologous cultured fibroblasts showed improvement whereas 0% of subjects treated with placebo showed improvement (P < 0.0001). Based on self-assessment at 24 weeks after the injection, 72% of subjects treated with autologous cultured fibroblasts and 45% of subjects treated with placebo showed improvement (P = 0.0662). There were no serious adverse events related to autologous cultured fibroblast injection., Conclusions: Autologous cultured fibroblast injection might be effective and safe to correct nasojugal grooves., Level of Evidence I: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Various types of skin substitutes composed of fibroblasts and/or keratinocytes have been used for the treatment of diabetic ulcers. However, the effects have generally not been very dramatic. Recently, human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have been commercialised for cartilage repair as a first cell therapy product using allogeneic stem cells. In a previous pilot study, we reported that hUCB-MSCs have a superior wound-healing capability compared with fibroblasts. The present study was designed to compare the treatment effect of hUCB-MSCs with that of fibroblasts on the diabetic wound healing in vitro. Diabetic fibroblasts were cocultured with healthy fibroblasts or hUCB-MSCs. Five groups were evaluated: group I, diabetic fibroblasts without coculture; groups II and III, diabetic fibroblasts cocultured with healthy fibroblasts or hUCB-MSCs; and groups IV and V, no cell cocultured with healthy fibroblasts or hUCB-MSCs. After a 3-day incubation, cell proliferation, collagen synthesis levels and glycosaminoglycan levels, which are the major contributing factors in wound healing, were measured. As a result, a hUCB-MSC-treated group showed higher cell proliferation, collagen synthesis and glycosaminoglycan level than a fibroblast-treated group. In particular, there were significant statistical differences in collagen synthesis and glycosaminoglycan levels (P = 0·029 and P = 0·019, respectively). In conclusion, these results demonstrate that hUCB-MSCs may have a superior effect to fibroblasts in stimulating diabetic wound healing., (© 2017 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Background: Capsular contracture is an incurable complication after silicone-based implant surgery. Myofibroblast is the predominant cell in the contracted capsule. We hypothesized that human adipose derive stromal cells (hASCs) together with fibroblast may show a similar phenotypic characteristics of myofibroblast after the treatment of inflammatory cytokines in vitro., Materials and Methods: Interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) were treated in the culture of hASCs and HDFs. Lyn peptide inhibitor was applied as an inhibitor. The changes of cell surface markers (CD105, CD73, CD34, CD45, CD31, CD325 and CD146) were assessed. The expression of various cytokines related to wound contraction were tested such as TGF-β, α-SMA, HGF, FGF, ENT-1, and TSP-1. Myo-D, α-SMA, and glial fibrillary acidic protein (GFAP) were evaluated by blotting and immunocytochemical staining. The collagen-gel contraction assay was performed for the functional contraction of myofibroblastic phenotype., Results: The expression of α-SMA, Myo-D and GFAP after the treatment of IL-3/GM-CSF showed similar results in hASCs and HDFs. Enhanced expression of TGF- β was observed in HDFs and the increase of ENT-1 and TSP-1 was significant in hASCs. Collagen-gel with HDFs contracted significantly within 24h after the treatment of IL-3/GM-CSF, and the contraction was inhibited by Lyn peptide inhibitor. But in hASCs, the gel-contraction was not significant., Conclusion: IL-3/ GM-CSF effected on the myofibroblastic differentiation of hASCs as well as it did on HDFs. But hASCs did not show the phenotypic gel-contraction within 24h., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Current studies of Panax ginseng (or Korean ginseng) have demonstrated that it has various biological effects, including angiogenesis, immunostimulation, antimicrobial and anti-inflammatory effects. Therefore, we hypothesised that P. ginseng may also play an important role in wound healing. However, few studies have been conducted on the wound-healing effects of P. ginseng. Thus, the purpose of this in vitro pilot study was to determine the effects of P. ginseng on the activities of fibroblasts, which are key wound-healing cells. Cultured human dermal fibroblasts were treated with one of six concentrations of P. ginseng: 0, 1, 10 and 100 ng/ml and 1 and 10 µg/ml. Cell proliferation was determined 3 days post-treatment using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, and collagen synthesis was evaluated by the collagen type I carboxy-terminal propeptide method. Cell proliferation levels and collagen synthesis were compared among the groups. The 10 ng/ml to 1 µg/ml P. ginseng treatments significantly increased cell proliferation, and the 1 ng/ml to 1 µg/ml concentrations significantly increased collagen synthesis. The maximum effects for both parameters were observed at 10 ng/ml. P. ginseng stimulated human dermal fibroblast proliferation and collagen synthesis at an optimal concentration of 10 ng/ml., (© 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

A new injectable tissue-engineered soft tissue consisting of a mixture of hyaluronic acid (HA) filler and cultured human fibroblasts have been developed by the authors. To establish this method as a standard treatment, a further study was required to determine whether the injected fibroblasts could stay at the injected place or move to other sites. In addition, effective strategies were needed to increase viability of the injected fibroblasts. The purpose of this study was to track the injected fibroblasts and to determine the effect of adding prostaglandin E1 (PGE1) or vitamin C on the viability of fibroblasts.Human fibroblasts labeled with fluorescence dye were suspended in HA filler and injected into 4 sites on the back of nude mice. The injected bioimplants consisted of one of the 4 followings: HA filler without cells (HA group), fibroblasts suspended in HA filler (HA + FB group), PGE1-supplemented fibroblasts in HA filler (HA + FB + PGE1 group), and vitamin C-supplemented fibroblasts in HA filler (HA + FB + VC group). At 4 weeks after injection, locations and intensities of the fluorescence signals were evaluated using a live imaging system.The fluorescence signals of the fibroblast-containing groups were visible only at the injected sites without dispersing to other sites. The HA +FB + PGE1 group showed a significantly higher fluorescence signal than the HA + FB and the HA + FB +VC groups (P < 0.05, each). There was no statistical difference between the HA + FB and HA + FB +VC groups (P = 0.69).The results of the current study collectively suggest that injected fibroblasts suspended in HA filler stay at the injected place without moving to other sites. In addition, PGE1 treatment may increase the remaining rhodamine B isothiocynanate dye at the injected site of the human dermal fibroblasts.

The authors previously reported that cultured human fibroblasts suspended in a hyaluronic acid filler can produce human dermal matrices with extended in vivo stability in animal and clinical studies. The present study was undertaken to determine the optimal viscosity and particle shape of hyaluronic acid filler as a scaffold for cultured human dermal fibroblasts to enhance the maximal viability of injected cells. The fibroblasts were suspended in either 1 of 3 hyaluronic acid viscosities at 2 different particle shapes. The viscosities used in this study were low (600,000-800,000 centipoises), moderate (2,000,000-4,000,000 centipoises), and high (8,000,000-12,000,000 centipoises). The particle shape was evaluated by testing round and irregular shapes. The fibroblast mixed bioimplants were injected into the back of individual athymic nude mice. The levels of type I collagen were measured using fluorescent-activated cell sorting (FACS) and immunohistochemical staining at 16 weeks after the injections. Results of FACS demonstrated that the mean cell ratio with human collagens in the moderate viscosity group was greater than those of control, low, and high viscosity groups. An immunohistochemical study showed similar results. The moderate viscosity group demonstrated the highest positive staining of human collagens. However, there were no significant differences between groups of irregular and round shape particles. A hyaluronic acid bioimplant with moderate viscosity is superior to that with low or high viscosity in the viability for human fibroblasts. However, the particle shape does not influence the viability of the fibroblasts.

In covering wounds, efforts should include use of the safest and least invasive methods with a goal of achieving optimal functional and cosmetic outcome. The recent development of advanced technology in wound healing has triggered the use of cells and/or biological dermis to improve wound healing conditions. The purpose of the study was to evaluate the effects of fibroblast-seeded artificial dermis on wound healing efficacy.Ten nude mice were used in this study. Four full-thickness 6-mm punch wounds were created on the dorsal surface of each mouse (total, 40 wounds). The wounds were randomly assigned to one of the following 4 treatments: topical application of Dulbecco phosphate-buffered saline (control), human fibroblasts (FB), artificial dermis (AD), and human fibroblast-seeded artificial dermis (AD with FB). On the 14th day after treatment, wound healing rate and wound contraction, which are the 2 main factors determining wound healing efficacy, were evaluated using a stereoimage optical topometer system, histomorphological analysis, and immunohistochemistry.The results of the stereoimage optical topometer system demonstrated that the FB group did not have significant influence on wound healing rate and wound contraction. The AD group showed reduced wound contraction, but wound healing was delayed. The AD with FB group showed decreased wound contraction without significantly delayed wound healing. Histomorphological analysis exhibited that more normal skin structure was regenerated in the AD with FB group. Immunohistochemistry demonstrated that the AD group and the AD with FB group produced less α-smooth muscle actin than the control group, but this was not shown in the FB group.Fibroblast-seeded artificial dermis may minimize wound contraction without significantly delaying wound healing in the treatment of skin and soft tissue defects.

Objective: Diabetic foot ulcers (DFUs) often pose a treatment problem. Bioengineered skin substitutes have been reported to result in accelerated diabetic wound healing. The purpose of this clinical trial was to evaluate the efficacy and safety of the autologous fibroblast-hyaluronic acid complex for treating DFUs., Method: A stratified, randomised, controlled, multicentre study was carried out. Patients with DFUs were allocated to either a treatment group with grafting of an autologous fibroblast-hyaluronic acid complex or a control group with non-adherent foam dressing. Except for application of the fibroblast complex, treatment of the study ulcers was identical for patients in both groups. The maximum follow-up period for each patient was 12 weeks., Results: Complete ulcer healing was achieved in 84% (26/31 patients) of the treatment group and 34% (11/32 patients) of the control group (p<0.05). The times required for complete healing were 36.4 ± 17.6 and 48.4 ± 13.1 days in the treatment and control groups, respectively (p<0.05). No adverse events related to treatment occurred., Conclusion: These results indicate that autologous fibroblast-hyaluronic acid complex may offer a safe and effective treatment for DFUs.

Cultured human fibroblasts have been used for soft-tissue augmentation in the face. However, if cultured cells are to be used clinically, the approval of the Food and Drug Administration and time-consuming culturing procedures are required. For this reason, we investigated adipose-derived stromal vascular fraction (SVF) cells, which could be obtained in large quantities without cell culture. However, unlike fibroblasts, SVF cells are a heterogenic cell population. Therefore, it was hypothesized that a SVF cell graft may possibly form various tissues. This study was performed to determine what tissue would form after a graft of SVF cells. Stromal vascular fraction cells were isolated from liposuctioned human adipose tissue. For a test group, SVF cells were suspended in hyaluronic acid filler. For a control group, no cells were included in the filler. These implants were subcutaneously injected into the backs of 4 nude mice. After 16 weeks, the injection areas were excised, and histologic examination was performed including immunohistochemistry to confirm the presence of human collagen. The test group specimens revealed fibrous connective tissues infiltrating into and surrounding the hyaluronic acids. Collagen fibers and fibroblasts were found around newly formed vascular structures. In contrast, the control group did not show cells or hyaluronic acid particles. Immunohistochemistry of the test group displayed strong expression of human collagen. However, the control group exhibited negative staining. This suggests that transplanted uncultured human SVF cells combined with hyaluronic acid filler generate fibrovascular tissue.

Human processed lipoaspirate (PLA) cells are relatively easy to obtain in large quantities without cell culture. The purpose of this study was to present the possibility of using uncultured PLA cell autografts for the treatment of diabetic ulcers. An in vitro study was designed initially to determine the effect of PLA cell autografts on the proliferation and collagen synthesis of diabetic fibroblasts (n=4). In a subsequent clinical study, 26 patients with diabetic foot ulcers were treated using PLA cell autografts. Control treatment was also performed in 26 patients. Eight weeks after treatment, the percentages of complete healing and mean healing times were compared. Cell proliferation and collagen synthesis in the PLA cell treatment group were 28 and 44% higher than that in the control group in vitro, respectively. Our clinical study showed that 100% of the PLA cell-treated group and 62% of the control group achieved complete healing. The PLA cell treatment was also superior to the conventional method in terms of the healing time. No adverse events related to the study treatment occurred. Uncultured PLA cell autografts stimulate the activity of diabetic fibroblasts and may offer a simple and effective treatment for diabetic ulcers.

In previous pilot studies, the authors reported that bone marrow stromal cells (BSCs) have a superior effect on healing of acute wounds compared with fibroblasts in vitro and in vivo. The present study was designed to compare the effect of allograft of normal BSCs with that of normal fibroblasts on the activity of diabetic fibroblasts in vitro. Diabetic fibroblasts were cultured in 24-well culture plates. Three groups were evaluated: group I, diabetic fibroblasts only; group II, diabetic fibroblasts co-cultured with normal fibroblasts; group III, diabetic fibroblasts co-cultured with normal BSCs. After 3-day incubation, cell proliferation and collagen synthesis, which are the major contributing factors in wound healing, were measured. Group III showed the highest level in both cell proliferation and collagen synthesis, followed by group II and group I (p<0.05). These results demonstrate that BSCs are superior to fibroblasts in stimulating the activity of diabetic fibroblasts., (Copyright 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Background and Objective: Various types of allogenic skin substitutes composed of cryopreserved keratinocytes, fibroblasts, or both have been used for treatments of diabetic foot ulcers, but the effects have generally not been dramatic because cryopreservation impairs cell activities. The purpose of the study was to evaluate the use of non-cryopreserved fresh human fibroblast allografts in treating diabetic foot ulcers., Materials and Methods: Human dermal fibroblasts from healthy teenagers were cultured and applied over the foot ulcers of 37 patients with diabetes. Control treatment was performed in 18 patients. Eight weeks after treatment, the percentages of complete healing, mean healing times, and patient satisfaction were compared, with follow-up ranging from 6 to 40 months., Results: Our study showed that 83.8% of the treated group and 50.0% of the control group experienced complete healing. The times required for complete healing were 30.9+/-10.1 and 47.2+/-7.8 days in the treated and control groups, respectively. Patient satisfaction with fresh fibroblast treatment was also superior to satisfaction with the conventional method (mean scores: 8.0+/-1.0 and 4.9+/-1.4, respectively). No adverse events related to the study treatment occurred., Conclusion: The use of fresh human fibroblast allografts was found to be a safe and effective treatment for diabetic foot ulcers. The authors have indicated no significant interest with commercial supporters.

In a previous in vitro study, the authors reported that bone marrow stromal cells (BSCs) have better wound-healing activities than fibroblasts. The purpose of this study was to evaluate the effect of BSCs and fibroblasts on wound-healing activity in vivo. Cultured human BSCs and dermal fibroblasts taken from the same patients were tested to compare collagen synthesis and epithelization in a rat wound model. No-cell-treated animals were used as controls. The BSC group showed the highest collagen level, followed by the fibroblast group, and then the no-cell group (P < 0.05). In addition, the best epithelization was observed in the BSC group. These results demonstrate that BSCs better stimulate wound healing than fibroblasts in vivo and in vitro.

Interleukin-1 beta (IL-1beta) is an abundant cytokine, which, together with TNF-alpha, mediates inflammatory events in rheumatoid arthritis (RA). IL-1beta is known to induce the induction of inflammatory cytokines and metalloproteinases (MMPs) in rheumatoid synovial cells. Here, we assessed these inflammatory events by measuring IL-1beta levels in the human synovial cell line, MH7A. We observed that the activation of p38 MAP kinase by IL-1beta was involved in the induction of inflammatory cytokines, as well as several genes, including MMP-1 and MMP-3. SB203580, a specific p38 MAP kinase inhibitor, inhibited the production of IL-1beta-induced cytokines and MMPs, while the levels of the tissue inhibitor of metalloproteinase (TIMPs) were unchanged by treatment with SB203580. Moreover, the induction of suppressor of cytokine signaling 3 (SOCS3) and interferon regulatory factor 1 (IRF-1) were both found to be induced by the inhibition of p38 MAP kinase. Therefore, we suggested that the inhibition of p38 MAP kinase might enhance anti-inflammatory tendencies in the MH7A cells.

In a previous animal study, the authors reported that cultured human fibroblasts suspended in Restylane, which is a modified hyaluronic acid, can produce human dermal matrices with extended in vivo stability. This study was undertaken to evaluate the clinical efficacy of this method, particularly for augmentation rhinoplasty cases. Between January 2002 and June 2003, 11 patients were treated with subcutaneous implants of Restylane mixed with autologous fibroblasts for augmentation rhinoplasty. Of these 11 patients, a long-term follow-up for more than 1 year was possible in 6 patients. Appearance of reconstructed noses, degree and time of resorption, occurrence of complications, and patients' satisfaction were investigated. The injected implants remained in situ without evidence of significant resorption or loss of correction. All patients were satisfied with the achieved long-term results, and no complication occurred. The results obtained indicate that this method is well tolerated and may have a potential to be an effective means of performing augmentation rhinoplasty.

Background: A cell therapy methodology for angiogenesis using fibroblasts has already been developed. Bone marrow stromal cells, which contain mesenchymal stem cells, have a low rate of immunity-assisted rejection and are capable of expanding profoundly in culture. Therefore, these cells offer several advantages for transplantation over mature cells. The aim of this study was to compare the angiogenic activity of bone marrow stromal cells with that of fibroblasts., Methods: For in vitro study, cultured human bone marrow stromal cells and dermal fibroblasts were seeded onto 96-well culture plates. After 1, 3, and 5 days, the levels of the basic fibroblast growth factor and vascular endothelial growth factor were compared. For in vivo study, porous polyethylene disks were loaded with bone marrow stromal cells, fibroblasts, or no cells and then implanted in the backs of rats. At three time intervals ranging from 1 to 3 weeks, the microvascular density was measured., Results: In the in vitro study, the basic fibroblast growth factor levels in the bone marrow stromal cell group were 47, 89, and 68 percent higher than in the fibroblast group at each time interval (p < 0.05). The vascular endothelial growth factor levels of the bone marrow stromal cell group were seven, 12, and 12 times higher than those of the fibroblast group (p < 0.05). In the in vivo study, there was little difference in the microvascular density among the three groups by the second week. However, the 3-week specimens showed a significantly greater difference. The microvascular density averaged 52.88, 26.12, and 17.50 for the bone marrow stromal cell, fibroblast, and no-cell groups, respectively., Conclusion: These results suggest that bone marrow stromal cells may possibly be used as a replacement for fibroblasts for angiogenesis.

To investigate the effect of basic Fibroblast Growth Factor (bFGF) on fibrovascular ingrowth into porous polyethylene orbital implants (Medpor) and to investigate any differences according to the method of administration. For the treated groups, after evisceration and Medpor implantation, bFGF was administered by soaking Medpor in the bFGF solution, and/or by injecting bFGF into the Medpor 1 week after the operation. Implants were removed 4 weeks after the operation and examined for the degrees of fibrovascular ingrowth by light microscopy. The percentages of the cross-sectional area of the implant occupied by fibrovascular ingrowth and the numbers of proliferated vessels were significantly higher in the bFGF-treated groups (Mann Whitney test, p<0.05). Administration routes had no effect on the degree of fibrovascular ingrowth (Kruskal-Wallis test, p>0.05). bFGF promoted fibrovascular ingrowth into porous polyethylene orbital implants regardless of the route of administration. Therefore, bFGF might be helpful to prevent complications such as implant exposure.

Diabetic foot ulcers often pose a difficult problem for health care professionals because of the defects associated with fibroblast functioning. Although there has been much interest recently in the use of topical growth factors for the treatment of diabetic foot ulcers, the effects are generally not very dramatic. Cryopreserved fibroblast implants, which are able to adjust to a wound's environment and provide the desired growth factors and other substances that may be lacking in a chronic wound, represent an exciting development and a major advance. These products may well provide growth factors in the right concentration and in the right sequence, something that has proved difficult to achieve with the topical application of recombinant growth factors. However, cell activities are impaired by cryopreservation. The purpose of this study was to assess the effects of fresh human allogeneic fibroblast grafting for the treatment of diabetic foot ulcers. Eight patients with diabetic foot ulcers ranging from 6 to 17 weeks in duration were treated. The size of the wounds ranged from 2.0 to 6.0 cm2, with three patients exhibiting exposed bones. A history of diabetic foot ulcers was present in five patients. Human dermal fibroblasts from healthy teenagers were cultured in Dulbecco's modified Eagle medium/Ham's F-12 supplemented with 10% autologous serum. The cultured cells were applied over the wounds immediately after debridement; fibrin was used as a cell carrier. A dressing was then applied with Tegaderm and kept moist until healing was complete. The progress and time for complete wound closure and patient satisfaction were assessed, with follow-up time ranging from 6 to 18 months. Complete wound healing occurred in all patients. Eleven to 21 days were needed for complete reepithelization of the wound, and no clinical or laboratory abnormalities were noted. Patient satisfaction was also very positive. In this study, the use of fresh human fibroblast allografts was found to be a safe and effective treatment for diabetic foot ulcers.

For the elimination of facial wrinkles and skin contour defects, injectable filler substances composed of commercially prepared nonanimal stabilized hyaluronic acid (Restylane) are now widely used. Although this method of suspension has been shown to be relatively safe and convenient, varying degrees of resorption have required repeated percutaneous injections. This study was undertaken to evaluate the feasibility of Restylane, which is a modified hyaluronic acid, combined with cultured human dermal fibroblasts, to enhance the longevity of injected implants. The histologic changes of the injected implants were also evaluated. For the test group, fibroblasts from the dermis of healthy adults were isolated and cultivated. The cultured fibroblasts were measured with a hemocytometer. Five x 105 fibroblasts suspended in 200 microl of Dulbecco phosphate-buffered saline (DPBS) were then dispersed in 200 microl of Restylane to form a 400-microl human fibroblast-Restylane mix. For the control group, 200 microl of DPBS without fibroblasts were mixed with 200 microl of Restylane. These implants were subcutaneously injected into the back of an athymic nude mouse at 6 sites, the 3 left sites composing the control group and the 3 right sites composing the test group. Twelve nude mice were injected for a total of 36 injections per group. The nodular swellings that resulted from the injections were excised to include skin beyond the swelling points down to the panniculus carnosus layer using 5-mm punches, and the weights were measured at 1, 2, 4, 8, 12, and 16 weeks after the injections. Histologic comparisons were also performed to confirm the presence of human collagen in the fibroblast-mixed Restylane group using immunohistochemical study with antihuman collagen type I polyclonal antibody. The mean weight of the control group nodules decreased throughout the examination period. The mean weight at the 16th week was 60% of the weight at the first week. On the other hand, the mean weight of the test-group nodules decreased only over the first 2 weeks. Beyond 2 weeks, there was no further significant weight change. The mean weight at the 16th week was 91% of the weight measured at the first week. Histologic examinations of the control group exhibited negative immunohistochemical staining for human collagen at each examination period. The test group exhibited positive staining after 2 weeks, indicating the presence of human collagen. These results indicate that Restylane mixed with cultured human dermal fibroblasts may be successfully injected as living grafts for long-term retention of implants.

Previously, we demonstrated that the gastrin releasing peptide (GRP) induces cyclooxygenase-2 (COX-2) expression through a Rho-dependent, protein kinase C (PKC)-independent signaling pathway in fibroblasts (Slice et al., 1999, J Biol Chem 274:27562-27566). However, the specific role of heterotrimeric guanine nucleotide binding regulatory proteins (G-proteins) that are coupled to the GRP receptor in Rho-dependent COX-2 expression has not been elucidated. In this report, we utilize embryonic fibroblasts from transgenic mice containing double gene knock-outs (DKO) for Galpha(q/11) and Galpha(12/13) to demonstrate that COX-2 promoter activation by GRP requires Galpha(q). Furthermore, we show that GRP-dependent COX-2 gene expression, as assessed by a COX-2 reporter luciferase assay, was induced in cells lacking Galpha(12/13) but was blocked in cells that did not express Galpha(q/11). GRP-dependent COX-2 promoter induction in Galpha(q/11) deficient cells was rescued by expression of wild type Galpha(q) but blocked by inhibition of calcium signaling in calcium-free media or in cells treated with 2-aminoethoxydiphenylborate (2-APB). Co-stimulation of transfected Galpha(q/11) deficient cells with GRP and thapsigargin (TG) induced the COX-2 promoter. Activation of endogenous Rho by expression of Onco-lbc or expression of Rho A Q63L resulted in COX-2 promoter activation in Galpha(q/11) deficient cells. Inhibition of Rho by Clostridium botulinum C3 toxin blocked COX-2 promoter induction. Expression of Galpha(q) Q209L in the well-characterized fibroblast cell line, NIH3T3, induced the COX-2 promoter which was blocked by expression of C3 toxin. These results demonstrate that calcium signaling mediated by Galpha(q) and Rho play critical roles in GRP-dependent COX-2 expression in fibroblasts., (Copyright 2002 Wiley-Liss, Inc.)