Allogeneic Dermal Fibroblasts Improve Tendon-to-Bone Healing in a Rabbit Model of Chronic Rotator Cuff Tear Compared With Platelet-Rich Plasma.

PURPOSE: To compare the effects of allogeneic dermal fibroblasts (ADFs) and platelet-rich plasma (PRP) on tendon-to-bone healing in a rabbit model of chronic rotator cuff tear. METHODS: Thirty-two rabbits were divided into 4 groups (8 per group). In 2 groups, the supraspinatus tendon was detached and was left as such for 6 weeks. At 6 weeks after creating the tear model, we performed transosseous repair with 5 × 106 ADFs plus fibrin injection in the left shoulder and PRP plus fibrin in the right shoulder. The relative expression of the COL1, COL3, BMP2, SCX, SOX9, and ACAN genes was assessed at 4 weeks (group A) and 12 weeks (group B) after repair. Histologic and biomechanical evaluations of tendon-to-bone healing at 12 weeks were performed with ADF injection in both shoulders in group C and PRP injection in group D. RESULTS: At 4 weeks, COL1 and BMP2 messenger RNA expression was higher in ADF-injected shoulders (1.6 ± 0.8 and 1.0 ± 0.3, respectively) than in PRP-injected shoulders (1.0 ± 0.3 and 0.6 ± 0.3, respectively) (P = .019 and P = .013, respectively); there were no differences in all genes in ADF- and PRP-injected shoulders at 12 weeks (P > .05). Collagen continuity, orientation, and maturation of the tendon-to-bone interface were better in group C than in group D (P = .024, P = .012, and P = .013, respectively) at 12 weeks, and mean load to failure was 37.4 ± 6.2 N/kg and 24.4 ± 5.2 N/kg in group C and group D, respectively (P = .015). CONCLUSIONS: ADFs caused higher COL1 and BMP2 expression than PRP at 4 weeks and showed better histologic and biomechanical findings at 12 weeks after rotator cuff repair of the rabbit model. ADFs enhanced healing better than PRP in the rabbit model. CLINICAL RELEVANCE: This study could serve as a transitional study to show the effectiveness of ADFs in achieving tendon-to-bone healing after repair of chronic rotator cuff tears in humans.

Application of Cultured Epidermal Homograft (Kaloderm) for Wide Scar Treatment.

BACKGROUND: For the treatment of wide scars, laser resurfacing procedures are generally used. However, sometimes their results are not satisfactory. Many clinical studies have reported that cultured epidermal allogenic sheets promote rapid and good quality wound healing. Therefore, the authors applied a cultured epidermal homograft (CEH) for scar management and investigated its outcomes. METHODS: Thirty-two patients who received a CEH (Kaloderm) after laser resurfacing (n = 14, under general anesthesia; n = 18, under local anesthesia) between February 2016 and June 2017 were enrolled. Patients treated with dermabrasion using laser resurfacing (n = 60) without CEH in the same period were used as controls. Clinical grading of the scars was performed using a Patient and Observer Scar Assessment Scale (POSAS) at postoperative 12 months. RESULTS: The authors conducted a comparative analysis between the control and CEH groups. Evaluation based on Patient and Observer Scar Assessment Scale showed that the mean scores in control/CEH groups for the 7 observer components (vascularity, pigmentation, thickness, relief, pliability, surface area, and overall opinion) were 4.5/3.2, 3.3/2.8, 2.8/2.5, 3.6/3.5, 3.7/2.1, 2.3/1.9, and 3.2/2.7, respectively, with significant differences observed in vascularity, pliability, and surface area (P values = 0.033, 0.021, and 0.048, respectively). Meanwhile, the mean scores in control/CEH groups for 7 patient components (pain, itching sense, color, stiffness, thickness, irregularity, and overall opinion) were 4.1/2.3, 3/3.1, 2.2/2.1, 2.2/1.7, 3.6/3.5, 1.8/1.5, and 2.2/1.9, respectively, with significant differences between groups observed in pain, stiffness, and overall opinion in the paired t test (P values = 0.041, 0.020, and 0.048, respectively). CONCLUSION: Cultured epidermal homograft provided good quality wound healing and improved scar pliability. Cultured epidermal homograft left less scarring with no pain or other specific complications. Therefore, dermabrasion with CEH is useful for scar management.

The Effect of Combining Hyaluronic Acid and Human Dermal Fibroblasts on Tendon Healing.

BACKGROUND: The incidence of rotator cuff tears is rapidly increasing, and operative techniques for rotator cuff repair have been developed. However, the rates of postoperative retear remain high. PURPOSE/HYPOTHESIS: The purpose was to determine the effects of human dermal fibroblasts (HDFs) with hyaluronic acid (HA) on tendon-to-bone healing in a rabbit model of chronic rotator cuff tear injury. It was hypothesized that HA would enhance HDF proliferation and that a combination of HA and HDFs would produce a synergistic effect on the healing of repaired rotator cuff tendons of rabbits. STUDY DESIGN: Controlled laboratory study. METHODS: For in vitro study, HDFs were plated on a 24-well plate. After 1 day, 2 wells were designated as the test group and treated with 0.75% HA in phenol red-free Dulbecco's modified Eagle medium (DMEM). An other 2 wells served as control groups and were treated with the same volume of phenol red-free DMEM without HA. Each group was duplicated, resulting in a total of 4 wells, with 2 wells in each group for replication purposes. The cells were incubated for 24 hours, followed by 72-hour cultivation. Absorbance ratios at 96 and 24 hours were compared to evaluate cell proliferation. For the in vivo study, a total of 24 rabbits were randomly allocated to groups A, B, and C (n = 8 each). Supraspinatus tendons were detached bilaterally and left for 6 weeks to establish a chronic rotator tear model. Torn tendons were subsequently repaired using the following injections: group A, 0.5 × 106 HDFs with HA; group B, HA only; and group C, saline only. At 12 weeks after repair, biomechanical tests and histological evaluation were performed. RESULTS: In vitro study showed that HDF proliferation significantly increased with HA (HDFs with HA vs HDFs without HA; 3.96 ± 0.09 vs 2.53 ± 0.15; P < .01). In vivo, group A showed significantly higher load-to-failure values than the other groups (53.8 ± 6.9 N/kg for group A, 30.6 ± 6.4 N/kg for group B, and 24.3 ± 7.6 N/kg for group C; P < .001). Histological evaluation confirmed that group A showed higher collagen fiber density and better collagen fiber continuity, tendon-to-bone interface maturation, and nuclear shape than the other groups (all P < .05). CONCLUSION: This controlled laboratory study verified the potential of the combination of HDFs and HA in enhancing healing in a chronic rotator cuff tear rabbit model. CLINICAL RELEVANCE: A potential synergistic effect on rotator cuff tendon healing may be expected from a combination of HDFs and HA.

Safety and Efficacy of Autologous Dermal Fibroblast Injection to Enhance Healing After Full-Thickness Rotator Cuff Repair: First-in-Human Pilot Study.

BACKGROUND: There is growing interest in various biological supplements to improve tendon healing in patients after arthroscopic rotator cuff repair. The ideal biological supplement to strengthen rotator cuff remains unknown. PURPOSE: To assess the safety and efficacy of autologous cultured dermal fibroblast (ADF) injection on tendon-to-bone healing in patients after arthroscopic rotator cuff repair. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Included were 6 patients who underwent arthroscopic rotator cuff repair between June 2018 and March 2020; all patients had a full-thickness rotator cuff tear (>2 cm) involving the supraspinatus and infraspinatus tendons. The patients were injected with ADF between the repaired tendon and footprint during arthroscopic rotator cuff repair using the suture bridge technique. The safety of ADFs and the procedure was evaluated at 5 weeks postoperatively, and the anatomical healing of the repaired tendon was accessed at 6 months postoperatively using magnetic resonance imaging and at 12 months using ultrasonography. Outcomes including shoulder range of motion (ROM), visual analog scale (VAS) for pain, and functional scores were measured at 6 and 12 months postoperatively. RESULTS: Adverse reactions to ADF injection were not observed at 6 months after surgery. VAS and functional scores at 6 and 12 months postoperatively were significantly improved compared with preoperative scores (all P < .05). However, there was no significant difference on any ROM variable between preoperative and postoperative measurements at 6 and 12 months (all P > .05). No healing failure was found at 6 and 12 months postoperatively. CONCLUSION: There was no adverse reaction to ADF injection, and all patients had successful healing after rotator cuff repair. A simple and easily accessible ADF injection may be a novel treatment option for increasing the healing capacity of torn rotator cuff tendons. Further clinical research is needed to verify the study results.

Rotator Cuff Tendon Healing Using Human Dermal Fibroblasts: Histological and Biomechanical Analyses in a Rabbit Model of Chronic Rotator Cuff Tears.

BACKGROUND: Tenocytes derived from tendons have been reported to be effective in the treatment of rotator cuff tears through the expression of extracellular matrix proteins. Human dermal fibroblasts, known to express collagen types I and III as tenocytes do, may likely be substitutes for tenocytes to enhance healing rotator cuff tears. PURPOSE: To demonstrate the capability of human dermal fibroblasts to enhance healing of rotator cuff tears. STUDY DESIGN: Controlled laboratory study. METHODS: The cellular properties and expression profiles of growth factors were compared between human dermal fibroblasts and tenocytes. In both cell types, a series of extracellular matrix proteins were analyzed along with matrix metalloproteinases and tissue inhibitors of metalloproteinases involved in the collagenolytic system. A total of 35 rabbits were divided into 5 groups: normal (n = 2), saline control (n = 9), fibrin control (n = 9), low dose of human fibroblasts (HF-LD; n = 9), and high dose of human fibroblasts (HF-HD; n = 6). Cells were injected into the sutured lesions at 6 weeks after creation of bilateral rotator cuff tears, followed by histological and biomechanical analyses at 12 weeks. RESULTS: Human dermal fibroblasts exhibited a protein expression pattern similar to that of tenocytes. More specifically, the expression levels of collagen types I and III were comparable between fibroblasts and tenocytes. The histological analysis of 30 surviving rabbits showed that collagen fibers were more continuous and better oriented with a more mature interface between the tendon and bone in the sutured lesions in the HF-LD and HF-HD groups. Most importantly, biomechanical strength, measured using the load to failure at the injection site, was 58.8 ± 8.9 N/kg in the HF-HD group, increasing by approximately 2-fold (P = .0003) over the saline control group. CONCLUSION: Human dermal fibroblasts, showing cellular properties comparable with tenocytes, effectively enhanced healing of chronic rotator cuff tears in rabbits. CLINICAL RELEVANCE: Human dermal fibroblasts can be used in place of tenocytes to enhance healing of rotator cuff tears.

Effects of Allogenic Dermal Fibroblasts on Rotator Cuff Healing in a Rabbit Model of Chronic Tear.

BACKGROUND: The failure of rotator cuffs to heal after repair is an unresolved surgical issue. There have been substantial efforts, including the use of biological supplements, to enhance tendon healing. Dermal fibroblasts are a good candidate for tendon tissue engineering because they are similar to the tenocytes used for collagen synthesis. In addition, they are easily accessible because autologous dermal fibroblasts can be obtained from individual skin without major skin defects and allogenic dermal fibroblasts (ADFs) have already been commercialized in the field of skin engineering. PURPOSE: To determine the effects of dermal fibroblasts on tendon-to-bone healing in a rabbit model of a chronic rotator cuff tear. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 33 rabbits were randomly allocated into 3 groups (n = 11 each). Supraspinatus tendons were detached and left for 6 weeks to establish a chronic rotator tear model. Torn tendons were repaired in a transosseous manner with the injection of 5 × 106 ADFs with fibrin in group A, fibrin only in group B, and saline only in group C. At 12 weeks after repair, the mechanical test and histological evaluation were performed. RESULTS: Seven rabbits died before the evaluation (1 in group A, 2 in group B, 4 in group C). In the final evaluation, the mean ± SD load to failure was 48.1 ± 13.3 N/kg for group A, 34.5 ± 8.9 N/kg for group B, and 31.1 ± 8.3 N/kg for group C, and group A showed significantly higher load-to-failure values than the other groups ( P = .011). The midsubstance tear rate, which presented stronger tendon-to-bone healing than insertional tear, was 50.0% in group A, 22.2% in group B, 28.6% in group C, but the differences were not statistically significant ( P = .413). In the histological evaluation, group A showed greater collagen fiber continuity and better orientation than the other groups. CONCLUSION: This controlled laboratory study verified, on the basis of biomechanics and histology, the potential for the use of ADFs in rotator cuff healing. The current results suggest a new biological supplement to increase the rate of rotator cuff healing. CLINICAL RELEVANCE: The most important finding of this study was the potential for a new biological supplement to enhance rotator cuff healing-a continuing challenge.

Prospective Clinical Trial of Corneal Reconstruction With Biomaterial-Free Cultured Oral Mucosal Epithelial Cell Sheets.

PURPOSE: To investigate the efficacy and safety of transplantation with biomaterial-free cultured oral mucosal epithelial cell sheets (COMECs) for ocular reconstruction in subjects with total limbal stem cell deficiency. METHODS: A prospective clinical trial (NCT02149732) was conducted in 8 subjects with total limbal stem cell deficiency after approval from the institutional review board of Seoul National University Hospital (H-0707-043-213) and the Ministry of Food and Drug Safety of Korea. COMECs were prepared in a culture system without the use of any temperature-sensitive polymers or carriers. The COMECs were transplanted without suture fixation. Four subjects underwent penetrating keratoplasty after stabilization of the COMEC transplant. Stable epithelialization, changes in visual acuity, and postoperative complications were evaluated for 6 months. Corneal cytokeratins (K) of 4 subjects who underwent penetrating keratoplasty were stained with an immunofluorescent agent. RESULTS: The ocular surface was successfully reconstructed in 6 eyes. Complete stable epithelialization was achieved within a mean of 53.6 days. Visual improvement (≥2 lines) was achieved in 62.5% of the eyes. K12 (corneal phenotype), K4, and K13 (mucosal phenotype) were well expressed in grafts after keratoplasty, whereas K1, K8, and K19 were barely expressed. No ocular infections, local tumor formation, or remarkable systemic complications were observed. Ocular reconstruction using COMECs failed in 2 eyes, which had full symblepharon in 4 quadrants. CONCLUSIONS: Transplanting biomaterial-free COMECs seems to be an efficient and safe procedure to reconstruct the ocular surface in patients who are completely limbal stem cell deficient without a full symblepharon.

Characterization of biomaterial-free cell sheets cultured from human oral mucosal epithelial cells.

The purpose of this study was to report the characteristics of biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal-deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony-forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki-67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial-free sheets. Biomaterial-free sheets which had been detached with 1% dispase or biomaterial-free sheets generated by fibrin support were transplanted to 12 limbal-deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial-free sheets were compared with those of sheets formed by fibrin-coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial-free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial-free sheets were comparable to those of sheets cultured by fibrin support, in limbal-deficient models. Both p63 and Ki-67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal-deficient models. Our results suggest that biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal-deficient diseases. Copyright © 2014 John Wiley & Sons, Ltd.

Comparative Analysis of Substrate-Free Cultured Oral Mucosal Epithelial Cell Sheets from Cells of Subjects with and without Stevens-Johnson Syndrome for Use in Ocular Surface Reconstruction.

PURPOSE: To compare the regenerative potential of cultured oral mucosal epithelial cells sheets (COMECs) from Stevens-Johnson syndrome (SJS) subjects with those from non-SJS subjects. METHODS: Human oral mucosal epithelial cells from SJS and non-SJS subjects were cultured, and colony-forming efficiency (CFE), proliferative and migration potential, expression of cytokines/growth factors and stem cells were compared. COMECs from SJS and non-SJS subjects were transplanted into 12 limbal stem cell-deficient rabbits, and their regenerative potential was analyzed at 1 week after transplantation. RESULTS: CFE (p>0.05, student's t test), cell proliferation potential (p>0.05, two-way ANOVA) and expression of the cytokeratins (K3, K4, K13, K19) in the oral mucosal epithelial cells from SJS subjects were similar to those of the cells from non-SJS subjects. The initial migratory potential of SJS cells was delayed compared to that of non-SJS cells (p <0.05, RM two-way ANOVA). The SJS cells expressed lower levels of EGF and higher levels of VEGF compared to that of non-SJS cells (p<0.05, one-way ANOVA). In vivo transplanted SJS-COMECs showed similar expression of K3, K4, and K13, proliferation markers (Ki-67; p>0.05, Mann-Whitney U test), and stem cell markers (p63; p>0.05, Mann-Whitney U test) compared to non-SJS COMECs. The initial epithelial defects in vivo were larger in the eyes treated with SJS-COMECs on day 3 (p<0.01, RM two-way ANOVA), but no differences were observed by day 7 between SJS- and non-SJS-COMECs. CONCLUSIONS: These results suggest that, aside from differences in migratory potential, oral mucosal epithelial cells from SJS and non-SJS subjects are comparable in their regeneration potential in treating limbal stem cell deficiency.

The control of cell adhesion and detachment on thin films of thermoresponsive poly[(N-isopropylacrylamide)-r-((3-(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)ammonium hydroxide)].

This paper describes the formation of poly(N-isopropylacrylamide) (PNIPAAm) and poly[(N-isopropylacrylamide)-r-((3-(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)ammonium hydroxide)] (P(NIPAAm-r-MPDSAH)) films on a glass surface via surface-initiated, atom transfer radical polymerization as a cell-culture platform. The films of PNIPAAm with various thicknesses and of P(NIPAAm-r-MPDSAH) with various ratios of NIPAAm and MPDSAH are formed to investigate the behaviors of cell adhesion and detachment. In the case of the PNIPAAm-grafted glass surfaces, the optimal film thickness, achieving the effective control of both cell adhesion and detachment, is estimated to be 11-13 nm for NIH 3T3 fibroblast cells. The adhesion and detachment behaviors of NIH 3T3 fibroblast cells are further tuned by incorporating the hydrophilic and non-biofouling MPDSAH moiety into the PNIPAAm system. The cell adhesion and detachment are controlled best, when the ratio of NIPAAm and MPDSAH is 75:1 (NIPAAm:MPDSAH).

Novel synthetic ceramide derivatives increase intracellular calcium levels and promote epidermal keratinocyte differentiation.

Ceramide is an important constituent of stratum corneum lipids, which act as both physical barriers and signal modulators. We synthesized several ceramide derivatives and investigated their effects on keratinocyte differentiation. RT-PCR and Western blotting showed that the novel synthetic ceramide derivatives K6PC-4 [N-(2,3-dihydroxypropyl)-2-hexyl-3-oxo-decanamide], K6PC-5, [N-(1,3-dihydroxypropyl-2-hexyl-3-oxo-decanamide] and K6PC-9 (N-ethanol-2-hexyl-3-oxo-decanamide) [corrected] These ceramide derivatives elicited a rapid transient increase in intracellular calcium levels, which were measured using laser scanning confocal microscopy. In addition, K6PC-4, K6PC-5, and K6PC-9 stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In a reconstituted epidermis model, K6PC-4, K6PC-5, and K6PC-9 significantly increased keratin 1 expression in the suprabasal layer. These results indicate that these novel synthetic ceramide derivatives have the potential to promote keratinocyte differentiation, suggesting that the lipid molecules are applicable for treating skin diseases involving abnormal keratinocyte differentiation.