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Bone regeneration remains a critical challenge in orthopedic medicine. Extracellular matrix proteins play important roles in bionic mineralization and osteogenesis. Sacchachitin nanofibers (SCNFs) oxidized via TEMPO have shown great potential as biomaterial scaffolds due to the introduction of carboxylic groups that mimic the natural extracellular matrix. However, TEMPO is harmful to the environment, which calls for greener and sustainable alternatives. Therefore, this study explored the green fabrication of oxidized-SCNFs through SiliaCat-TEMPO (STEMPO) as a renewable catalyst with various amount of oxidation agents to evaluate their bone regeneration ability. We developed an optimal STEMPO oxidation process, enabling efficient removal of most STEMPO particles through centrifugation with minimal residue. The oxidation efficiency of recovered STEMPO particles remained comparable to fresh ones. STEMPO-oxidized SCNFs, oxidized at increasing NaClO levels, showed an increase in carboxylate content, from 0.1360 mmol/g to 3.2415 mmol/g. Additionally, STEMPO-oxidized SCNFs exhibited excellent biocompatibility with MC3T3-E1 osteoblastic cells, facilitating calcium precipitation, cell migration, and osteogenic differentiation. Mixed composites of β-tricalcium phosphate (BCP) and STEMPO-oxidized SCNFs with higher oxidation levels further enhanced osteogenic differentiation. In vivo studies using a rat femur defect model showed that SCNFs at the highest oxidation level (ST100SC) significantly promoted bone regeneration to full recovery, achieving a bone volume to total tissue volume (BV/TV) ratio of 75.6 %, compared to 48.3 % in the control. These results demonstrated that STEMPO-oxidized SCNFs with higher oxidation levels can be used as a bioactive matrix scaffold to achieve full recovery of bone regeneration.

In this study, an ultrasound-assisted digestion method of a formic acid-decellularized extracellular matrix (dECM) of porcine skin was developed and optimized to form UdECM hydrogels for diabetic wound healing. Results demonstrated that ultrasonication improved the extraction rate of collagen from dECM samples, preserved the collagen content of dECM, reduced residual cells, and extracted greater DNA contents. Scanning electron microscope (SEM) analyses were performed, which demonstrated the optimal porosity on the surface and density of the cross-section in the hydrogel structure, which could control the release of growth factors embedded in UdECM hydrogels at desirable rates to boost wound healing. A wound-healing study was conducted with six different composite hydrogels, both empty materials and materials enriched with rat platelet-rich plasma (R-PRP), sacchachitin nanofibers (SCNFs), and TEMPO-oxidized sacchachitin in diabetic rats. The assessment based on scars stained with hematoxylin and eosin (H&E), Masson's trichrome (MT), and a cluster of differentiation 31 (CD31) staining showed that the UdECM/SC/R-PRP treatment group had the most significant efficacy of promoting healing and even recovery of diabetic wounds to normal tissues. UdECM/R-PRP and UdECM/SCNFs demonstrated better healing rates than UdECM hydrogel scaffolds, which had only recovered 50% resemblance to normal skin. Treatment with both UdECM/TEMPO 050 and UdECM/TEMPO 050/R-PRP hydrogel scaffolds was ranked last, with even poorer efficacy than UdECM hydrogels. In summary, formulated UdECM and SCNF hydrogels loaded with PRP showed synergistic effects of accelerating wound healing and ultimately stimulating the wound to recover as functional tissues. This newly UdECM/SCNF composite hydrogel has promising potential for healing and regenerating diabetic wounds. [ABSTRACT FROM AUTHOR]

In this study, an ultrasound-assisted digestion method of a formic acid-decellularized extracellular matrix (dECM) of porcine skin was developed and optimized to form UdECM hydrogels for diabetic wound healing. Results demonstrated that ultrasonication improved the extraction rate of collagen from dECM samples, preserved the collagen content of dECM, reduced residual cells, and extracted greater DNA contents. Scanning electron microscope (SEM) analyses were performed, which demonstrated the optimal porosity on the surface and density of the cross-section in the hydrogel structure, which could control the release of growth factors embedded in UdECM hydrogels at desirable rates to boost wound healing. A wound-healing study was conducted with six different composite hydrogels, both empty materials and materials enriched with rat platelet-rich plasma (R-PRP), sacchachitin nanofibers (SCNFs), and TEMPO-oxidized sacchachitin in diabetic rats. The assessment based on scars stained with hematoxylin and eosin (H&E), Masson’s trichrome (MT), and a cluster of differentiation 31 (CD31) staining showed that the UdECM/SC/R-PRP treatment group had the most significant efficacy of promoting healing and even recovery of diabetic wounds to normal tissues. UdECM/R-PRP and UdECM/SCNFs demonstrated better healing rates than UdECM hydrogel scaffolds, which had only recovered 50% resemblance to normal skin. Treatment with both UdECM/TEMPO 050 and UdECM/TEMPO 050/R-PRP hydrogel scaffolds was ranked last, with even poorer efficacy than UdECM hydrogels. In summary, formulated UdECM and SCNF hydrogels loaded with PRP showed synergistic effects of accelerating wound healing and ultimately stimulating the wound to recover as functional tissues. This newly UdECM/SCNF composite hydrogel has promising potential for healing and regenerating diabetic wounds.

Abstract Background Ganoderma sp., such as Ganoderma tsugae (GT), play an important role in traditional Chinese medicine. Ganoderma sp. contains several constituents, including Sacacchin, which has recently drawn attention because it can not only enhance the repair of muscle damage but also strengthen the muscle enforcement. Although Ganoderma sp. have a therapeutic effect for neuromuscular disorders, the underlying mechanism remains unclear. This study investigated the effect and underlying molecular mechanism of micronized sacchachitin (mSC) on satellite cells (SCs), which are known as the muscle stem cells. Methods The myogenic cells, included SCs (Pax7+) were isolated from tibialis anterior muscles of a healthy rat and were cultured in growth media with different mSC concentrations. For the evaluation of SC proliferation, these cultivated cells were immunostained with Pax7 and bromodeoxyuridine assessed simultaneously. The molecular signal pathway was further investigated by using Western blotting and signal pathway inhibitors. Results Our data revealed that 200 µg/mL mSC had an optimal capability to significantly enhance the SC proliferation. Furthermore, this enhancement of SC proliferation was verified to be involved with activation of TAK1-JNK-AP-1 signaling pathway through TLR2, whose expression on SC surface was confirmed for the first time here. Conclusion Micronized sacchachitin extracted from GT was capable of promoting the proliferation of SC under a correct concentration.

Hong-Liang Lin,1 Ting-Huan Wu,2 Hsiu-O Ho,2 Fang-Ching Chao,2 Meng-Huang Wu,3,4 Der-Zen Liu,5 Ling-Chun Chen,6 Ming-Thau Sheu2 1School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China; 2School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan, Republic of China; 3Department of Orthopedics, Taipei Medical University Hospital, Taipei, Taiwan, Republic of China; 4Department of Orthopedics, College of Medicine, Taipei Medical University, Taipei, Taiwan, Republic of China; 5Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan, Republic of China; 6Department of Biotechnology and Pharmaceutical Technology, Yuanpei University of Medical Technology, Hsinchu, Taiwan, Republic of ChinaCorrespondence: Ling-Chun Chen; Ming-Thau SheuTaipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan, Republic of ChinaTel +886-2-27361661 ext. 6112Fax +886-2-23771942Email d8801004@tmu.edu.tw; mingsheu@tmu.edu.twIntroduction: In this study, the combination of TEMPO-oxidized sacchachitin nanofibers (TOSCNFs) with chitosan-activated platelet-rich plasma (cPRP) was evaluated for remedying dry eye syndrome (DES).Methods: TOSCNFs, designated T050SC, were generated. T050SC combined with chitosan-activated (cPRP) was formulated as eye drops for application for severe DES. To evaluate the effects of cPRP and TOSCNFs on the repair of corneal injury, in vitro studies were conducted using Statens Seruminstitut rabbit corneal (SIRC) epithelial cells for cell proliferation and cell migration assays, and a severe DES animal model using rabbits was established with benzalkonium chloride (BAC) treatment for the evaluation.Results: Results showed that the optimal eye formulation contained PRP activated by 350 μg/mL of the low-molecular-weight chitosan group (L3) combined with 300 μg/mL TO50SC (L3+T050SC). In the WST-1 cell-proliferation assay, L3 and L3+TO50SC significantly increased Statens SIRC cell proliferation after 24 hrs of incubation. In the SIRC cell migration assay, the L3+TO50SC group showed a wound-healing efficiency of 89% after 24-hr treatment. After 5 days of treatment, Schirmer’s test results did not simulate the dry eye animal model. Typical cornea appearance and eye fluorescein staining results showed that the L3 group had the best effect on improving cornea haze and epithelial damage.Conclusion: This study has determined that TOSCNFs effectively promoted the healing effect on severe cases of corneal damage, and also might enhance the clinical application and medical potential of PRP in ophthalmology.Keywords: TEMPO-oxidation, sacchachitin, PRP, dry eye syndrome, nanofibers

Extracellular matrix (ECM) hydrogel can create a favorable regenerative microenvironment and act as a promising dressing for accelerating the healing of diabetic wound. In this study, a simple and effective decellularization technique was developed and optimized to obtain acellular extracellular matrix (aECM) from porcine skin. It was found that decellularization at 30% formic acid for 72 h effectively decellularized porcine skin while retaining >75% collagen and ~37% GAG in the aECM with no presence of nuclei of cellular remnants. aECM hydrogel was fabricated by digesting aECM with pepsin in various acidic solutions (0.1 N HCl, glycolic acid (GA) and 2-pyrrolidone-5-carboxylic acid (PCA)) and then treated with a pH-controlled neutralization and temperature-controlled gelation procedure. Based on physical characterizations, including SDS-PAGE, rheological analysis and SEM analysis, aECMHCl hydrogels fabricated at 25 mg/mL in 0.1 N HCl were selected. Four polymeric ECM-mimic hydrogels, including sacchachitin (SC), hyaluronic acid (HA) and chitosan (CS) and three composite hydrogels of combining SC either with aECMHCl,25 (aECMHCl/SC), HA (HA/SC) or CS (SC/CS) were prepared and evaluated for WS-1 cell viability and wound-healing effectiveness. Cell viability study confirmed that no hydrogel dressings possessed any toxicity at all concentrations examined and ECMHCl, HA and ECMHCl/SC at higher concentrations (>0.05%) induced statistically significant proliferation. Diabetic wound healing study and histological examinations revealed that ECMHCl/SC hydrogel was observed to synergistically accelerate wound healing and ultimately stimulated the growth of hair follicles and sweat glands in the healing wound indicating the wound had healed as functional tissues. The results support the great potential of this newly produced ECMHCl/SC composite hydrogel for healing and regeneration of diabetic wounds.

Microbial control plays an important role in the clinical efficacy of dental practice. The purpose of this study was to develop a long-lasting antibacterial guided-tissue regeneration (GTR) barrier containing the non-shellfish chitosan from the fruiting body residue of Ganoderma tsugae. The powder of ground up G. tsugae fruiting body residue was processed to prepare the chitosan. The molecular weight, degree of deacetylation (DD), and yield of these non-shellfish chitosan derivatives were characterized. This material was also combined with other biomaterials to prepare polylactic acid (PLA)IChitosanl amorphous calcium phosphate (ACP) GTR barrier films for periodontal applications. By washing with deionized water, these GTR films were coagulated from the lactic acid solution. The film morphology was investigated using scanning electron microscopy, and the antibacterial function was also evaluated. The yields and DDs of the sacchachitin and chitosan were 39% ± 1% and 1% ± 0.2%, and 25% ± 2% and 68% ± 2%, respectively. Tests for chitosan against Actinobacillus actinomycetemcomitans showed that the antibacterial ability of the GTR barrier films, containing 2.08 wt% of the resultant chitosan, was 2.25 times higher, compared to 5 wt% doxycycline after the 8th day. The GTR barrier film maintained 56.58% of its antibacterial ability after the 18th day. We concluded that sacchachitin and chitosan from G. tsugae fruiting body residues possess excellent, long-lasting antibacterial function for potential dental applications. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) hydrogel can create a favorable regenerative microenvironment and act as a promising dressing for accelerating the healing of diabetic wound. In this study, a simple and effective decellularization technique was developed and optimized to obtain acellular extracellular matrix (aECM) from porcine skin. It was found that decellularization at 30% formic acid for 72 h effectively decellularized porcine skin while retaining >75% collagen and ~37% GAG in the aECM with no presence of nuclei of cellular remnants. aECM hydrogel was fabricated by digesting aECM with pepsin in various acidic solutions (0.1 N HCl, glycolic acid (GA) and 2-pyrrolidone-5-carboxylic acid (PCA)) and then treated with a pH-controlled neutralization and temperature-controlled gelation procedure. Based on physical characterizations, including SDS-PAGE, rheological analysis and SEM analysis, aECMHCl hydrogels fabricated at 25 mg/mL in 0.1 N HCl were selected. Four polymeric ECM-mimic hydrogels, including sacchachitin (SC), hyaluronic acid (HA) and chitosan (CS) and three composite hydrogels of combining SC either with aECMHCl,25 (aECMHCl/SC), HA (HA/SC) or CS (SC/CS) were prepared and evaluated for WS-1 cell viability and wound-healing effectiveness. Cell viability study confirmed that no hydrogel dressings possessed any toxicity at all concentrations examined and ECMHCl, HA and ECMHCl/SC at higher concentrations (>0.05%) induced statistically significant proliferation. Diabetic wound healing study and histological examinations revealed that ECMHCl/SC hydrogel was observed to synergistically accelerate wound healing and ultimately stimulated the growth of hair follicles and sweat glands in the healing wound indicating the wound had healed as functional tissues. The results support the great potential of this newly produced ECMHCl/SC composite hydrogel for healing and regeneration of diabetic wounds. [ABSTRACT FROM AUTHOR]

• TEMPO-oxidized SCNF (TOSCNF) and microfludized SCNF (MDSCNF) were prepared and evalued. • All 2% MDSCNF suspension were demonstrated to be in a gel form. • All except T100SC of 2% TOSCNF suspension showed to be wet fiber-like hydrogel. • SCN5/H and T050SC/H accelerated diabetic wound healing to similar as normal tissue. • T050SC/H provided the healed wound as functional tissue instead of scar tissue. TEMPO-oxidization and mechanical disintegration were utilized to develop sacchachitin nanofibers (SCNF) with a 3D gel structure for being an ideal scaffold. Mechanically disintegrated SCNF (MDSCNF) with NanoLyzer® at 20,000 psi for 5 cycles and TEMPO-oxidized SCNF (TOSCNF) produced with 5.0 and 10.0 mmole NaClO/g SC was designated as SCN5, T050SC, and T100SC, respectively. All 2% MDSCNF suspensions were demonstrated to be in gel form, while all except T100SC of 2% TOSCNF suspensions showed to be wet fiber-like hydrogel. In diabetic wound healing study, both SCN5 and T050SC incorporated in AMPS (2-acrylamide-2-methyl-propane sulfonate)-based wound dressing were showed to accelerate diabetic wound healing forming nearly the same as normal tissues. T050SC/H further provided the healed wound with growth of sweat glands and hair follicles indicating the wound had healed as functional tissue. Conclusively, TEMPO-oxidized SCNF-based hydrogel scaffolds showed greater potentials in tissue regeneration due to its unique physical and chemical properties. [ABSTRACT FROM AUTHOR]

The extraction residue of the Ganoderma fruiting body, named sacchachitin, has been demonstrated to have the potential to enhance cutaneous wound healing by inducing cell proliferation. In this study, a nanogel formed from micronized sacchachitin (mSC) was investigated for the potential treatment of superficial chemical corneal burns. Reportedly, mSC has been produced successfully and its chemical properties confirmed, and physical and rheological properties characterized. An in vitro cell proliferation study has revealed that at the concentrations of 200, 300, and 400 microg/mL, mSC nanogel significantly increased Statens Seruminstitut rabbit corneal (SIRC) cell proliferation after 24 hours of incubation. In cell migration assay, migration of SIRC cell to wound closure was observed after 24 hours of incubation with the addition of 200 microg/mL mSC of nanogel. In an animal study, acceleration of corneal wound healing was probably due to the inhibition of proteolysis. In conclusion, the findings of this study substantiate the potential application of sacchachitin in the form of mSC nanogel for the treatment of superficial corneal injuries.