Impact of Thermo-Responsive N-Acetylcysteine Hydrogel on Dermal Wound Healing and Oral Ulcer Regeneration.

This study investigates the efficacy of a thermo-responsive N-acetylcysteine (NAC) hydrogel on wound healing and oral ulcer recovery. Formulated by combining NAC with methylcellulose, the hydrogel's properties were assessed for temperature-induced gelation and cell viability using human fibroblast cells. In vivo experiments on Sprague Dawley rats compared the hydrogel's effects against saline, NAC solution, and a commercial NAC product. Results show that a 5% NAC and 1% methylcellulose solution exhibited optimal outcomes. While modest improvements in wound healing were observed, significant enhancements were noted in oral ulcer recovery, with histological analyses indicating fully regenerated mucosal tissue. The study concludes that modifying viscosity enhances NAC retention, facilitating tissue regeneration. These findings support previous research on the beneficial effects of antioxidant application on damaged tissues, suggesting the potential of NAC hydrogels in improving wound care and oral ulcer treatment.

Oral ulcer treatment using human tonsil-derived mesenchymal stem cells encapsulated in trimethyl chitosan hydrogel: an animal model study.

BACKGROUND: Oral ulcers are a common side effect of chemotherapy and affect patients' quality of life. While stem cell transplantation is a potential treatment for oral ulcers, its efficacy is limited as the stem cells tend to remain in the affected area for a short time. This study aims to develop a treatment for oral ulcers by using trimethyl chitosan (TMC) hydrogel with human tonsil-derived stem cells (hTMSCs) to increase the therapeutic effect of stem cells and investigate their effectiveness. METHODS: Animals were divided into four experimental groups: Control, TMC hydrogel, hTMSCs, and hTMSCs loaded in TMC hydrogel (Hydrogel + hTMSCs) (each n = 8). Oral ulcers were chemically induced by anesthetizing the rats followed by injection of dilute acetic acid in the right buccal mucosa. After confirming the presence of oral ulcers in the animals, a single subcutaneous injection of 100 µL of each treatment was applied to the ulcer area. Histological analyses were performed to measure inflammatory cells, oral mucosal thickness, and fibrosis levels. The expression level of inflammatory cytokines was also measured using RT-PCR to gauge therapeutic the effect. RESULTS: The ulcer size was significantly reduced in the TMC hydrogel + hTMSCs group compared to the control group. The stem cells in the tissue were only observed until Day 3 in the hTMSCs treated group, while the injected stem cells in the TMC Hydrogel + hTMSCs group were still present until day 7. Cytokine analysis related to the inflammatory response in the tissue confirmed that the TMC Hydrogel + hTMSCs treated group demonstrated superior wound healing compared to other experimental groups. CONCLUSION: This study has shown that the adhesion and viability of current stem cell therapies can be resolved by utilizing a hydrogel prepared with TMC and combining it with hTMSCs. The combined treatment can promote rapid healing of oral cavity wounds by enhancing anti-inflammatory effects and expediting wound healing. Therefore, hTMSC loaded in TMC hydrogel was the most effective wound-healing approach among all four treatment groups prolonging stem cell survival. However, further research is necessary to minimize the initial inflammatory response of biomaterials and assess the safety and long-term effects for potential clinical applications.

Antiseptic, Hemostatic, and Wound Activity of Poly(vinylpyrrolidone)-Iodine Gel with Trimethyl Chitosan.

Wound management practices have made significant advancements, yet the search for improved antiseptics persists. In our pursuit of solutions that not only prevent infections but also address broader aspects of wound care, we investigated the impact of integrating trimethyl chitosan (TMC) into a widely used poly(vinylpyrrolidone)-iodine gel (PVP-I gel). Our study assessed the antimicrobial efficacy of the PVP gel with TMC against Escherichia coli, Staphylococcus aureus, multidrug-resistant S. aureus MRSA, and Candida albicans. Additionally, we compared hemostatic effects using a liver puncture bleeding model and evaluated wound healing through histological sections from full-thickness dermal wounds in rats. The results indicate that incorporating TMC into the commercially available PVP-I gel did not compromise its antimicrobial activity. The incorporation of TMC into the PVP-I gel markedly improves its hemostatic activity. The regular application of the PVP-I gel with TMC resulted in an increased blood vessel count in the wound bed and facilitated the development of thicker fibrous tissue with a regenerated epidermal layer. These findings suggest that TMC contributes not only to antimicrobial activity but also to the intricate processes of tissue regeneration. In conclusion, incorporating TMC proves beneficial, making it a valuable additive to commercially available antiseptic agents.

Photobiomodulation Recovers the Submandibular Gland in Vismodegib-Treated Rats.

Objective: The submandibular gland (SMG) produces the most saliva, and factors such as aging and chemotherapy can affect its structure and function. However, there are only temporary treatments available for salivary hypofunction. This study aimed to evaluate the effects of photobiomodulation (PBM) on the function of SMG by using a rat animal model and vismodegib, an antagonist of the sonic hedgehog (SHH) pathway. Methods: Vismodegib (10 mg/kg) drug was gavaged orally for 14 days in rats to significantly decrease the SHH signaling proteins [SHH, protein patched homolog 1 (PTCH1), smoothened protein (SMO), glioma-associated oncogene homolog 1 (GLI1)], induce damage in SMG tissue, and affect salivary functional markers AQP5 and Keratin5. After that, in conjunction with vismodegib administration, PBM was performed using an 850 nm high-power light-emitting diode (LED) device treated daily for 6 days at varying total energy densities of 60, 120, and 180 J/cm2 in at least 3 rats per group. The test results were confirmed by Western blot, immunofluorescence staining, and hematoxylin and eosin staining, and the statistics were t-test or one-way analysis of variance (ANOVA) with Tukey's multiple comparisons tests. Results: Significant decreases in the expression of SHH-related proteins (PTCH1, SMO, GLI1, p < 0.05) with damage of SMG ductal cells were observed with vismodegib administration. However, a significant increase in the expression levels of SHH-related proteins (SHH, SMO, GLI1, p < 0.05) and recovery of SMG ductal cells damaged after vismodegib administration were observed for PBM-treated groups. Salivary functional marker AQP5 also showed the same increase or decrease. Conclusions: This study found that vismodegib damages SMG ductal cells and decreases SHH-related proteins and associated salivary functional markers. Also, 850 nm high-power LED recovered the damaged structure of SMG and increased SHH-related proteins and salivary functional markers. The study results suggest that PBM can restore SMG structure and function through SHH signaling.

Photobiomodulation enhances M2 macrophage polarization properties of tonsil-derived mesenchymal stem cells.

In this study, the effect of photobiomodulation (PBM) treatment using 630 nm light emitting diode (LED) array (continuous wave type, 10 mW power) on tonsil-derived mesenchymal stem cells (TMSCs) and its interaction with RAW 264.7 macrophage cells via co-culture in vitro were investigated. PBM treatment was used as a priming method for TMSCs to improve therapeutic efficacy. TMSCs were subjected to multi-dose PBM treatments before co-culture with M1 activated (1 µg/mL lipopolysaccharide, LPS) macrophage cells with total energy doses of 0, 15, 30, and 60 J. Irradiation set at 15 J (1500 s treatment time) was performed once, twice for 30 J, and four times for 60 J in an incubator kept at 37 °C and 5% CO2. No significant anti-inflammatory response was observed for TMSCs co-cultured with macrophage cells without PBM. But PBM treatment of TMSCs with 630 nm LED array at 30 J reduced expression of inducible nitric oxide synthase, iNOS (M1) and increased expression of Arginase-1, Arg-1 (M2) phenotype macrophage markers. Anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RA) gene expression also increased significantly. Based on the results, PBM priming of TMSCs supports M2 macrophage polarization. PBM can be used to improve the therapeutic efficacy of TMSCs for potential applications in oral mucositis and wound healing.

Regeneration of taste through sonic hedgehog upregulation by photobiomodulation.

This study investigated photobiomodulation (PBM) effects in Sonic hedgehog (Shh) signaling as a potential approach to taste preservation and regeneration. Primary taste cell (TC) cultures were treated with Shh antagonist vismodegib and irradiated using a continuous wave type 630 nm light-emitting diode (10 mW/cm2 ) array, with single or multiple doses of 30 J/cm2 to determine dose inducing significant upregulation effect. Shh, Ptch, Smo, and Gli1 were significantly upregulated at 120 J/cm2 , used as the minimum dose in vivo. Vismodegib was administered via daily oral gavage for 21 days (30 mg/kg) to induce Shh inhibition in the tongue of rat animal models resulting in taste bud damage and taste dysfunction. PBM treatment using a 630 nm laser (3 W/cm2 ) at a radiant exposure of 120 J/cm2 (24 J/cm2 × 5) successfully upregulated the Shh protein expression, regenerated taste buds, and recovered taste function.

Recovery of sweet taste preference in adult rats following bilateral chorda tympani nerve transection.

Background: Numerous studies have noted the effect of chorda tympani (CT) nerve transection on taste sensitivity yet very few have directly observed its effects on taste receptor and taste signaling protein expressions in the tongue tissue. Methods: In this study, bilateral CT nerve transection was performed in adult Sprague Dawley rats after establishing behavioral taste preference for sweet, bitter, and salty taste via short term two-bottle preference testing using a lickometer setup. Taste preference for all animals were subsequently monitored. The behavioral testing was paired with tissue sampling and protein expression analysis. Paired groups of CT nerve transected animals (CTX) and sham operated animals (SHAM) were sacrificed 7, 14, and 28 days post operation. Results: Immunofluorescence staining of extracted tongue tissues shows that CT nerve transection resulted in micro-anatomical changes akin to previous investigations. Among the three taste qualities tested, only the preference for sweet taste was drastically affected. Subsequent results of the short-term two-bottle preference test indicated recovery of sweet taste preference over the course of 28 days. This recovery could possibly be due to maintenance of T1R3, GNAT3, and TRPM5 proteins allowing adaptable recovery of sweet taste preference despite down-regulation of both T1R2 and Sonic hedgehog proteins in CTX animals. This study is the first known attempt to correlate the disruption in taste preference with the altered expression of taste receptors and taste signaling proteins in the tongue brought about by CT nerve transection.

Inclusion of calcium phosphate does not further improve in vitro and in vivo osteogenesis in a novel, highly biocompatible, mechanically stable and 3D printable polymer.

At a time of unpredictable challenges for health, one trend is certain: there is an exceedingly high demand for functional implants, particularly bone grafts. This has encouraged the emergence of bone tissue engineering substitutes as an alternative method to conventional bone grafts. However, the current approaches in the field face several limitations that have prevented the ultimate translation into clinical settings. As a result, many attempts have been made to fabricate synthetic bone implants that can offer suitable biological and mechanical properties.Light curable methacrylate-based polymers have ideal properties for bone repair. These materials are also suitable for 3D printing which can be applicable for restoration of both function and aesthetics. The main objective of this research was to investigate the role of calcium phosphate (CaP) incorporation in a mechanically stable, biologically functional and 3D printable polymer for the reconstruction of complex craniofacial defects. The experimental work initially involved the synthesis of (((((((((((3R,3aR,6S,6aR)- hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(ethane-2,1- 48 diyl))bis(oxy))bis(carbonyl))bis(azanediyl))bis(3,3,5-trimethylcyclohexane-5,1- 49 diyl))bis(azanediyl))bis(carbonyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) referred to as CSMA and fabrication of composite discs via a Digital Light Printing (DLP) method. The flow behaviour of the polymer as a function of CaP addition, surface remineralisation potential, in vitro cell culture, using MC3T3 and Adipose-Derived Mesenchymal Stem Cells (ADSCs) and ex ovo angiogenic response was assessed. Finally, in vivo studies were carried out to investigate neo-bone formation at 4- and 8-weeks post-implantation. Quantitative micro-CT and histological evaluation did not show a higher rate of bone formation in CaP filled CSMA composites compared to CSMA itself. Therefore, such polymeric systems hold promising features by allowing more flexibility in designing a 3D printed scaffold targeted at the reconstruction of maxillofacial defects.

Trimethyl chitosan postoperative irrigation solution modulates inflammatory cytokines related to adhesion formation.

Lavage or irrigation has been instilled in surgical practice for wound clearance and surgical site infection prevention during and after surgery. Herein, we developed a new irrigation solution using trimethyl chitosan (TMC), a quaternized chitosan derivative. The TMC-saline irrigation solution developed in the study possesses highly effective bactericidal properties with hemostatic and anti-adhesion properties. The anti-adhesion property of TMC was investigated in relation to inflammatory cytokine response and wound healing. TMC-saline irrigation solution showed reduced pro-inflammatory cytokine protein and gene expressions relevant in the cascade of wound healing and cytokine-related orchestration of postoperative adhesion formation. Further development of this multifunctional TMC-saline irrigation solution can be beneficial for surgical applications and postoperative wound management.

Improved healing and macrophage polarization in oral ulcers treated with photobiomodulation (PBM).

OBJECTIVES: The effect of photobiomodulation (PBM) treatment on wound healing and macrophage polarization was investigated in vivo. Animal models of oral ulcers were simulated through chemically induced oral ulcers in rats. MATERIALS AND METHODS: PBM treatment using an infrared pulsed laser was used to treat oral ulcers in the animal models. Twelve Sprague-Dawley rats were randomly divided into four groups depending on set absorbed energy: Group 1 (control), Group 2 (30 J), Group 3 (60 J), and Group 4 (100 J). Laser treatment was performed every other day for 8 days after ulcer confirmation. Parameters used were as follows: wavelength 808 nm, power output 50 mW, spot size 10 mm, frequency 10 Hz, and pulse duration 1 millisecond. Ulcers were measured to determine the effect of the treatments over time. Histology, immunostaining, and real-time polymerase chain reaction analyses were performed to evaluate the effect of PBM treatment on macrophage-related (IL-6/IL-10) and wound-healing-related (TNF-α/TGF-ß/MMP-2) cytokine expression. RESULTS: Histological examinations indicate that the PBM treatment stimulated a higher level of wound recovery after 8 days of treatment at 60 J absorbed energy compared to other treatment groups. Analyses of relative gene expression of proinflammatory, anti-inflammatory, and tissue remodeling cytokines indicate that the macrophages in the tissue samples were predominantly characterized as M2 subtypes (alternatively activated), which possibly accounts for the accelerated tissue repair in the animal model of oral ulcer. CONCLUSION: This preliminary study stands as a proof of concept regarding the potential use of infrared laser PBM treatment for oral ulcers which have not been previously investigated upon. PBM treatment affects macrophage polarization and enhances wound healing. Further experimentation will be conducted to expand the understanding of how PBM treatment affects the healing mechanism of ulcers.

Boosting osteogenic potential and bone regeneration by co-cultured cell derived extracellular matrix incorporated porous electrospun scaffold.

Implants for bone regeneration to remedy segmental bone defects, osteomyelitis, necrotic bone tissue and non-union fractures have worldwide appeal. Although biomaterials offer most of the advantages by improving tissue growth but developments are more commonly achieved via biologically derived molecules. To aid site specific bone tissue regeneration by synthetic scaffold, cell derived extracellular matrix (ECM) can be a crucial component. In this study, co-cultured bone marrow mesenchymal stem cell and osteoblastic cells derived ECM incorporated electrospun polycaprolactone (PCL) membranes were assessed for bone tissue engineering application. The preliminary experimental details indicated that, co-culture of cells supported enhanced in vitro ECM synthesis followed by successful deposition of osteoblastic ECM into electrospun membranes. The acellular samples revealed retention of ECM related biomacromolecules (collagen, glycosaminoglycan) and partial recovery of pores after decellularization. In vitro biocompatibility tests ensured improvement of proliferation and osteoblastic differentiation of MC3T3-E1 cells in decellularized ECM containing membrane (PCL-ECM) compared to bare membrane (PCL-B) which was further confirmed by osteogenic marker proteins expression analysis. The decellularized PCL-ECM membrane allowed great improvement of bone regeneration over the bare membrane (PCL-B) in 8 mm size critical sized rat skull defects at 2 months of post implantation. In short, the outcome of this study could be impactful in development and application of cell derived ECM based synthetic electrospun templates for bone tissue engineering application.[Formula: see text].

Comparative study on biodegradation and biocompatibility of multichannel calcium phosphate based bone substitutes.

The objective of this study was to fabricate multichannel biphasic calcium phosphate (BCP) and ß-tricalcium phosphate (TCP) bone substitutes and compare their long-term biodegradation and bone regeneration potentials. Multi-channel BCP and TCP scaffolds were fabricated by multi-pass extrusion process. Both scaffolds were cylindrical with a diameter of 1-mm, a length of 1-mm, and seven interconnected channels. Morphology, chemical composition, phase, porosity, compressive strength, ion release behavior, and in-vitro biocompatibility of both scaffolds were studied. In-vivo biodegradation and bone regeneration efficacies of BCP and TCP were also evaluated using a rabbit model for 1 week, 1 month, and 6 months. BCP exhibited superior compressive strength compared to TCP scaffold. TCP showed higher release of both calcium ions and phosphorous ions than BCP in SBF solution. Both scaffolds showed excellent in-vitro biocompatibility and upregulated the expression of osteogenic markers of MC3T3-E1 cells. In-vivo studies revealed that both cylindrical TCP and BCP scaffolds were osteoconductive and supported new bone formation. Micro-CT data showed that the bone-regeneration efficacy of TCP was higher at one month and at six months after implantation. Histological examination confirmed that TCP degraded faster and had better bone regeneration than BCP after 6 months.

Evaluation of bone regeneration potential of injectable extracellular matrix (ECM) from porcine dermis loaded with biphasic calcium phosphate (BCP) powder.

Extracellular matrix (ECM) contains a wide array of complex proteins, growth factors and cytokines that regulate cell behavior and tissue development. ECM harvested from non-homologous ECM sources still provide a structural support and biochemical cues to cells for effective tissue remodeling. The aim of this study is to evaluate the effect of non-tissue specific decellularized ECM from porcine dermis loaded with biphasic calcium phosphate powder (BCP) in bone regeneration. Thermosensitive ECM hydrogels with BCP powder exhibited a porous morphology with a suitable injectability and increased mechanical stability. In-vitro studies using MC3T3-E1 pre osteoblast cells showed that the injectable ECM hydrogels were biocompatible and supported the osteogenic differentiation. The bone regeneration capacity of the injectable ECM hydrogels was evaluated in-vivo by implanting in rat femoral head for 4 and 8 weeks. Micro-CT and histological staining results indicated that the injectable ECM hydrogels loaded with BCP powder showed higher and improved bone formation compared with the unfilled defect. Injectable ECM loaded with BCP powder is a good potential biomaterial for non-load bearing bone regeneration application.

Enhanced decellularization technique of porcine dermal ECM for tissue engineering applications.

Effective removal of cellular components while retaining extracellular matrix (ECM) proteins is the ultimate goal of decellularization. The aim of this study is to produce a decellularized ECM with highly preserved ECM proteins and to determine the effect of isopropanol as a decellularization solvent on the characteristics of the decellularized porcine skin. Two different protocols were used for porcine skin decellularization. Protocol 1 consisted of Triton-X and sodium dodecyl sulfate (SDS) in water while protocol 2 consisted of Triton-X and SDS in 70% isopropanol. After decellularization, DNA components decreased significantly in protocol 2 with lower amount of lipid content and higher ECM proteins such as collagen (92.91 ±â€¯9.02 µg/mg sample), α-elastin (142.32 ±â€¯6.74 µg/mg sample) and sulfated glycosaminoglycan (sGAG; 7.44 ±â€¯1.30 µg/mg sample) compared with protocol 1 ECM. Higher amount of vascular endothelial growth factor (VEGF; 11.26 ±â€¯0.44 pg/mg sample) content was quantified in protocol 2 compared with protocol 1 while higher trace amount of bone morphogenic protein 2 (BMP-2; 0.28 ±â€¯0.04 pg/mg sample) was also observed in protocol 2 compared with protocol 1. Protocol 2 ECM did not significantly affect the cell viability and exhibited no cytotoxicity when exposed to three different cell lines: L929 fibroblast cells, MC3T3-E1 pre-osteoblast cells, and rat mesenchymal stem cells (BMSC). Subcutaneous implantation after 7 and 21 days revealed higher cell infiltration in protocol 2 ECM and enhanced neovascularization. Isopropanol/surfactants proved to be effective in cell and lipid removal during decellularization while preserving the higher amount of ECM proteins.

In vitro and in vivo evaluation of bioglass microspheres incorporated brushite cement for bone regeneration.

Bioglass-calcium phosphate cement (CPC) composite materials have recently received increased attention for bone regeneration purposes, owing to their improved properties in term of biocompatibility and bone ingrowths. In this study, an injectable bone substitute (IBS) system which utilizes bioglass microspheres incorporated into brushite based cement, was evaluated. The microspheres were synthesized with a simple and low sintering temperature process; there was no significant phase difference shown from the powder and good interactivity with cells was obtained. Furthermore, physical properties were optimized in microsphere incorporated brushite cement in order to investigate in vitro and in vivo performance. Accordingly, setting time and compressive strength were hardly altered until a microsphere content of 40% (v/v) was reached. The brushite (BR)/bioglass microsphere (BM) system showed excellent bioactivity to the in-vitro simulated body fluid test: dissolution ions from composite materials influenced apatite growth, countered acidic pH, and increased material degradation. In an in-vitro study with preosteoblasts (MC3T3-E1), BR/BM supported cell adhesion and proliferation, while cell differentiation experiments without osteogenic supplements, demonstrated that BR/BM induced osteogenic differentiation. A post-implantation study conducted in femoral defects showed higher materials degradation and bone formation in BR/BM than in BR. The faster dissolution of bioglass microspheres increased BR/BM composite resorption and hence facilitated bone tissue integration. Our findings suggest that bioglass microspheres incorporated in cement could potentially be used as an injectable bone substitute for bone regeneration applications.

Preparation and evaluation of BCP-CSD-agarose composite microsphere for bone tissue engineering.

Composite microspheres have been widely investigated over the years in order to achieve a sound scaffold with suitable combinations of biodegradable polymers and bioactive ceramics/glasses for bone tissue engineering. In our present study, composite microspheres were prepared for the first time by agarose (1 wt %) enforcement with combination of biphasic calcium phosphate (BCP; 20 wt %) and calcium sulfate dehydrate (CSD; 20 wt %), and analyzed for use in bone regeneration. The one-step fabrication process revealed spheres of sizes ranging from 50 to 1000 µm of BCP-CSD contents effectively formed by natural solidification of agarose matrix, which is very simple, time and cost-effective, and could allow for large scale production. Furthermore, the BCP-CSD-agarose composite microspheres were tested in in vitro and in vivo for bone-forming properties in order to assess their biocompatibility. The rapid diffusion of Ca 2+ ions from CSD of the composite microspheres through agarose matrix potentially increased interactivity with microenvironment and gave support for cell adhesion and proliferation. Moreover, in vivo result demonstrated that fabricated microspheres promoted neovascularization, stimulated fibroblast cell proliferation, and host cell migration occurred throughout the defects and within microspheres, ultimately guided to new bone formation. The developed composite microspheres with novel approach could have potential for bone regeneration application. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2263-2272, 2019.

Hemostasis and Bone Regeneration Using Chitosan/Gelatin-BCP Bi-layer Composite Material.

The aim of the study was to determine the hemostatic activity of a composite bi-layered topical hemostat composed of electrospun gelatin loaded with bi-phasic calcium phosphate and chitosan layer and its effect on bone formation. Morphology of the composite hemostat and its individual components were observed using scanning electron microscopy. In vitro biocompatibility of the topical hemostat tested using preosteoblasts cells (MC3t3-E1) showed no adverse toxicity. Confocal microscopy of seeded cells showed good cell adhesion while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated 15% increased cell proliferation at the end of 1 week culture period. The material's efficiency as a hemostatic agent was tested by testing blood adsorption capacity and in vivo bone bleeding models. Blood absorption indicates that test sample measuring 14.14 mm becomes fully saturated within 5 minutes of blood contact. Bone bleeding was induced on the frontal plates of rat skulls and samples were applied as either removable topical hemostat or actual degradable hemostat. The effect of the bi-layered hemostat on bone formation was determined through analysis of micro-computed tomography (microCT) and observation of histological sections of extracted bone tissue samples. Results indicate that the bi-layer hemostat was able to halt bleeding within 3 minutes of application on the bleeding site and significantly enhanced bone regeneration. Using the bi-layer material as a degradable hemostat also drastically improved bone regeneration of the 3 mm defect.