BACKGROUND: The skin, a vital organ protecting against microorganisms and dehydration, undergoes structural decline with aging, leading to visible issues such as wrinkles and sagging. Reduced blood vessels exacerbate vulnerability, hindering optimal cellular function and compromising skin health. Polydioxanone (PDO) biomaterials address aging concerns but produce acidic byproducts, causing inflammation. Inorganic particles and nitric oxide (NO) play crucial roles in inhibiting inflammation and promoting skin regeneration. Stem cell-derived extracellular vesicles (EVs) contribute to intercellular communication, offering the potential to enhance cell functions. The study proposes a method to enhance PDO-based medical devices by incorporating inorganic particles and immobilizing EVs, focusing on facial rejuvenation, anti-inflammatory response, collagen formation, and angiogenesis.METHOD: PDO composites with inorganic particles such as magnesium hydroxide (MH) and zinc oxide (ZO) were prepared and followed by EV immobilization. Comprehensive characterization included biocompatibility, anti-inflammation, collagen formation ability, and angiogenesis ability. RESULTS: Bulk-modified PDO composites demonstrated even dispersion of inorganic particles, pH neutralization, and enhanced biocompatibility. EVs immobilized on the composite surface exhibited spherical morphology. Inflammationrelated gene expressions decreased, emphasizing anti-inflammatory effects. Collagen-related gene and protein expressions increased, showcasing collagen formation ability. In addition, angiogenic capabilities were notably improved, indicating potential for skin rejuvenation. CONCLUSION The study successfully developed and characterized PDO composites with inorganic particles and EVs, demonstrating promising attributes for medical applications. These composites exhibit biocompatibility, anti-inflammatory properties, collagen formation ability, and angiogenic potential, suggesting their utility in skin rejuvenation and tissue engineering. Further research and clinical validation are essential.

BACKGROUND: Collagen is a key component of connective tissue and has been frequently used in the fabrication of medical devices for tissue regeneration. Human-originated collagen is particularly appealing due to its low immune response as an allograft biomaterial compared to xenografts and its ability to accelerate the regeneration process. Ethically and economically, adipose tissues available from liposuction clinics are a good resource to obtain human collagen.However, studies are still scarce on the extraction and characterization of human collagen, which originates from adipose tissue. The aim of this study is to establish a novel and simple method to extract collagen from human adipose tissue, characterize the collagen, and compare it with commercial-grade porcine collagen for tissue engineering applications. METHODS: We developed a method to extract the collagen from human adipose tissue under quasi-Good Manufacturing Practice (GMP) conditions, including freezing the tissue, blood removal, and ethanol-based purification. Various techniques, including protein quantification, decellularization assessment, SDS-PAGE, FTIR, and CD spectroscopy analysis, were used for characterization. Amino acid composition was compared with commercial collagen. Biocompatibility and cell proliferation tests were performed, and in vitro tests using collagen sponge scaffolds were conducted with statistical analysis. RESULTS: Our results showed that this human adipose-derived collagen was equivalent in quality to commercially available porcine collagen. In vitro testing demonstrated high cell attachment and the promotion of cell proliferation. CONCLUSION In conclusion, we developed a simple and novel method to extract and characterize collagen and extracellular matrix from human adipose tissue, offering a potential alternative to animal-derived collagen for xeno-free tissue engineering applications.

BACKGROUND: Autologous fat grafting is one of the most common procedures used in plastic surgery to correct soft tissue deficiency or depression deformity. However, its clinical outcomes are often suboptimal, and lack of metabolic and architectural support at recipient sites affect fat survival leading to complications such as cyst formation, calcification.Extracellular matrix-based scaffolds, such as allograft adipose matrix (AAM) and poly(lactic-co-glycolic) acid (PLGA), have shown exceptional clinical promise as regenerative scaffolds. Magnesium hydroxide (MH), an alkaline ceramic, has attracted attention as a potential additive to improve biocompatibility. We attempted to combine fat graft with regenerative scaffolds and analyzed the changes and viability of injected fat graft in relation to the effects of injectable natural, and synthetic (PLGA/MH microsphere) biomaterials. METHODS: In vitro cell cytotoxicity, angiogenesis of the scaffolds, and wound healing were evaluated using human dermal fibroblast cells. Subcutaneous soft-tissue integration of harvested fat tissue was investigated in vivo in nude mouse with random fat transfer protocol Fat integrity and angiogenesis were identified by qRT-PCR and immunohistochemistry. RESULTS: In vitro cell cytotoxicity was not observed both in AAM and PLGA/MH with human dermal fibroblast.PLGA/MH and AAM showed excellent wound healing effect. in vivo, the AAM and PLGA/MH retained volume compared to that in the only fat group. And the PLGA/MH showed the highest angiogenesis and anti-inflammation. CONCLUSION In this study, a comparison of the volume retention effect and angiogenic ability between autologous fat grafting, injectable natural, and synthetic biomaterials will provide a reasonable basis for fat grafting.

BACKGROUND: In order to produce and isolate the exosome derived from the cell of interests, a serum free environment (starvation) has been essential for excluding the unknown effect from serum-derived exosomes. Recently, serum-free culture media have been developed as a substitute for serum supplemented media so that MSC proliferates with maintaining the original characteristics of the cells in a serum free condition. Due to the different properties of the exosomes representing the states and characteristics of the origin cells, a study is needed to compare the properties of the cell-derived exosomes according to the cell culture media. METHODS: To compare the cell culture condition on exosomes, human umbilical cord mesenchymal stem cells (UCMSCs) were cultured with two different media, serum containing media, 10% FBS supplemented DMEM (NM) and serum-free chemically defined media, CellCor TM CD MSC (CDM). To remove FBS-derived exosomes from UCMSC cultured with NM, the medium was replaced with FBS-free DMEM for starvation during exosome isolation. The production yield and expression levels of angiogenic and pro-inflammatory factors were compared. And, the subpopulations of exosome were classified depending on the surface properties and loaded cytokines. Finally, the wound healing and angiogenic effects have been evaluated using in vitro assays. RESULTS: The UCMSC-derived exosomes under two different cell culture media could be classified into subpopulations according to the surface composition and loaded cytokines. Especially, exosome derived from UCMSC cultured with CDM showed higher expression levels of cytokines related to regenerative bioactivities which resulted in enhanced wound healing and angiogenesis. CONCLUSION CDM has the advantages to maintain cell proliferation even during the period of exosome isolations and eliminate unknown side effects caused by serumderived exosomes. Additionally, exosomes derived from UCMSC cultured with CDM show better wound healing and angiogenic effects due to a lot of regeneration-related cytokines and less pro-inflammatory cytokines compared to with NM.

BACKGROUND: Three-dimensional (3D) in vitro cultures recapitulate the physiological microenvironment and exhibit high concordance with in vivo conditions. Improving co-culture models with different kind of cell types cultured on a 3D scaffold can closely mimic the in vivo environment. In this study, we examined the osteogenic response of pre-osteoblast MC3T3-E1 cells and Raw264.7 mouse monocytes in a 3D-encapsulated co-culture environment composed of the Cellrix®3D culture system, which provides a physiologically relevant environment. METHODS: The Cellrix® 3D Bio-Gel scaffolds were used to individually culture or co-culture two type cells in 3D microenvironment. Under 3D culture conditions, osteoblastic behavior was evaluated with an ALP assay and staining. ACP assay and TRAP staining were used as osteoclastic behavior indicator. RESULTS: Treatment with osteoblastic induction factors (?3F) and RANKL had on positively effect on alkaline phosphatase activity but significantly inhibited to acid phosphatase activity during osteoclastic differentiation in 3D coculture. Interestingly, alkaline phosphatase activity or acid phosphatase activity in 3D co-culture was stimulated with opposite differentiation factors at an early stage of differentiation. We guess that these effects may be related to RANK– RANKL signaling, which is important in osteoblast regulation of osteoclasts. CONCLUSION In this study, the osteogenic response of 3D encapsulated pre-osteoblast MC3T3-E1 cells and mouse monocyte Raw264.7 cells was successfully demonstrated. Our 3D culture conditions will be able to provide a foundation for developing a high-throughput in vitro bone model to study the effects of various drugs and other agents on molecular pathways.

Background: The purpose of this retrospective study was to evaluate the postoperative change in the position and stability of the mandibular condyle after bilateral sagittal split ramus osteotomy (BSSRO) and BSSRO with distal segmental ostectomy (DSO) in patients with facial asymmetry using 3D computed tomography. Methods: The condyles of the patient diagnosed with facial asymmetry were divided into the deviated side (DS) and the non-deviated side (NDS). Group I, which was treated with BSSRO only, and Group II, which additionally received DSO along with BSSRO, were superimposed on the condyle using the pre-and postoperative 3D CT. The amount of condylar change in anteroposterior displacement, mediolateral displacement, and rotation was measured. The clinical symptoms of temporomandibular joint were also evaluated before and after surgery for each patient. Results: Between Groups I and II, there was no statistically significant difference in the anteroposterior condylar position on both DS and NDS.And also, there was no statistical difference between the two groups in the mediolateral change on DS but, statistically significant difference on NDS. The change in the rotation of the condyle was observed to rotate inward from both condylar heads of Groups I and II, and a statistically significant difference was observed between the two groups on both DS and NDS. Moreover, no difference in clinical temporomandibular joint symptoms was observed after surgery in each DS and NDS condyle of the two groups. Conclusions As a result of analyzing the condylar position change of the group treated with BSSRO alone and the group treated with BSSRO and DSO in patients with facial asymmetry, there were statistically significant differences in the mediolateral displacement of NDS and the condyle rotation of NDS and DS. However, the anteroposterior condylar position did not show any difference in the bilateral condyles. In addition, since worsening clinical symptoms of bilateral temporomandibular joint were not observed before and after surgery in both groups, it is concluded that it is not necessary to accompany DSO in patients with facial asymmetry (minimum 3 mm, maximum 7 mm).

BACKGROUND: In order to produce and isolate the exosome derived from the cell of interests, a serum free environment (starvation) has been essential for excluding the unknown effect from serum-derived exosomes. Recently, serum-free culture media have been developed as a substitute for serum supplemented media so that MSC proliferates with maintaining the original characteristics of the cells in a serum free condition. Due to the different properties of the exosomes representing the states and characteristics of the origin cells, a study is needed to compare the properties of the cell-derived exosomes according to the cell culture media. METHODS: To compare the cell culture condition on exosomes, human umbilical cord mesenchymal stem cells (UCMSCs) were cultured with two different media, serum containing media, 10% FBS supplemented DMEM (NM) and serum-free chemically defined media, CellCor TM CD MSC (CDM). To remove FBS-derived exosomes from UCMSC cultured with NM, the medium was replaced with FBS-free DMEM for starvation during exosome isolation. The production yield and expression levels of angiogenic and pro-inflammatory factors were compared. And, the subpopulations of exosome were classified depending on the surface properties and loaded cytokines. Finally, the wound healing and angiogenic effects have been evaluated using in vitro assays. RESULTS: The UCMSC-derived exosomes under two different cell culture media could be classified into subpopulations according to the surface composition and loaded cytokines. Especially, exosome derived from UCMSC cultured with CDM showed higher expression levels of cytokines related to regenerative bioactivities which resulted in enhanced wound healing and angiogenesis. CONCLUSION CDM has the advantages to maintain cell proliferation even during the period of exosome isolations and eliminate unknown side effects caused by serumderived exosomes. Additionally, exosomes derived from UCMSC cultured with CDM show better wound healing and angiogenic effects due to a lot of regeneration-related cytokines and less pro-inflammatory cytokines compared to with NM.

BACKGROUND: Three-dimensional (3D) in vitro cultures recapitulate the physiological microenvironment and exhibit high concordance with in vivo conditions. Improving co-culture models with different kind of cell types cultured on a 3D scaffold can closely mimic the in vivo environment. In this study, we examined the osteogenic response of pre-osteoblast MC3T3-E1 cells and Raw264.7 mouse monocytes in a 3D-encapsulated co-culture environment composed of the Cellrix®3D culture system, which provides a physiologically relevant environment. METHODS: The Cellrix® 3D Bio-Gel scaffolds were used to individually culture or co-culture two type cells in 3D microenvironment. Under 3D culture conditions, osteoblastic behavior was evaluated with an ALP assay and staining. ACP assay and TRAP staining were used as osteoclastic behavior indicator. RESULTS: Treatment with osteoblastic induction factors (?3F) and RANKL had on positively effect on alkaline phosphatase activity but significantly inhibited to acid phosphatase activity during osteoclastic differentiation in 3D coculture. Interestingly, alkaline phosphatase activity or acid phosphatase activity in 3D co-culture was stimulated with opposite differentiation factors at an early stage of differentiation. We guess that these effects may be related to RANK– RANKL signaling, which is important in osteoblast regulation of osteoclasts. CONCLUSION In this study, the osteogenic response of 3D encapsulated pre-osteoblast MC3T3-E1 cells and mouse monocyte Raw264.7 cells was successfully demonstrated. Our 3D culture conditions will be able to provide a foundation for developing a high-throughput in vitro bone model to study the effects of various drugs and other agents on molecular pathways.

Background: Inflammation induces dysfunction of endothelial cells via inflammatory cell adhesion, and this phenomenon and reactive oxygen species accumulation are pivotal triggers for atherosclerosis-related vascular disease. Although exosomes are excellent candidate as an inhibitor in the inflammation pathway, it is necessary to develop exosome-mimetic nanovesicles (NVs) due to limitations of extremely low release rate and difficult isolation of natural exosomes. NVs are produced in much larger quantities than natural exosomes, but due to the low flexibility of the cell membranes, the high loss caused by hanging on the filter membranes during extrusion remains a challenge to overcome. Therefore, by making cell membranes more flexible, more efficient production of NVs can be expected. Methods: To increase the flexibility of the cell membranes, the suspension of umbilical cord-mesenchymal stem cells (UC-MSCs) was subjected to 5 freeze and thaw cycles (FT) before serial extrusion. After serial extrusion through membranes with three different pore sizes, FT/NVs were isolated using a tangential flow filtration (TFF) system. NVs or FT/NVs were pretreated to the human coronary artery endothelial cells (HCAECs), and then inflammation was induced using tumor necrosis factor-α (TNF-α). Results: With the freeze and thaw process, the production yield of exosome-mimetic nanovesicles (FT/NVs) was about 3 times higher than the conventional production method. The FT/NVs have similar biological properties as NVs for attenuating TNF-α induced inflammation. Conclusion We proposed the efficient protocol for the production of NVs with UC-MSCs using the combination of freeze and thaw process with a TFF system. The FT/NVs successfully attenuated the TNF-α induced inflammation in HCAECs.