Adipose Collagen Fragment: A Novel Adipose-Derived Extracellular Matrix Concentrate for Skin Filling

Background Skin filler is an option for treatment of skin aging and wrinkle formation; however, currently used fillers are limited by poor biocompatibility, rapid degradation, and possible hypersensitivity reactions. However, autologous adipose tissue-derived products have been recognized as promising options for skin rejuvenation. Objectives This study aimed to develop a novel adipose-derived product for skin filling. Methods Adipose collagen fragment (ACF) was prepared through pulverization, filtration, and centrifugation. The macrography, structure, types of collagen, and cell viability of ACF were evaluated by immunostaining, Western blotting, and cell culture assays. ACF, nanofat and phosphate-buffered saline (9 spots/side, 0.01 ml/spot) were intradermally injected in the dorsal skin of 36 female BALB/c nude mice; then, the skin filling capacity and collagen remodeling process were investigated. Twenty-one female patients with fine rhytides in the infraorbital areas were enrolled and received ACF treatment as clinical applications. Therapeutic effects and patients’ satisfaction scores were recorded. Results The ACF yield from 50 ml of Coleman fat was 4.91 ± 0.25 ml. ACF contained nonviable cells and high levels of collagen I, collagen IV, and laminin. Fibroblasts and procollagen significantly increased in ACF and ACF-treated dermis (p < 0.05). 85.7% of patients were satisfied with the therapy results, and no infections, injection site nodules, or other unwanted side effects were observed. Conclusions ACF significantly improved dermal thickness and collagen synthesis and may serve as a potential autologous skin filler.

An Adipose-Derived Injectable Sustained-Release Collagen Scaffold of Adipokines Prepared Through a Fast Mechanical Processing Technique for Preventing Skin Photoaging in Mice

Ultraviolet A (UVA) radiation is the major contributor to skin photoaging, associated with increased collagen degradation and reactive oxygen species (ROS) expression. Adipokines have been proven as promising therapeutic agents for skin photoaging. However, adipokine therapy is generally limited by the short in vivo release duration and biological instability. Therefore, developing a treatment that provides a sustained release of adipokines and enhanced therapeutic effects is desirable. In this study, we developed a novel mechanical processing technique to extract adipose tissue-derived ECM components, named the “adipose collagen fragment” (ACF). The physical characterization, injectability, collagen components, residual DNA/RNA and adipokine release pattern of ACF were identified in vitro. L929 cells were treated with ACF or phosphate-buffered saline for 24 h after UVA irradiation in vitro. The expression of senescence-associated xβ-galactosidase (SA-β-gal), ROS and antioxidase were investigated. Then, we evaluated its therapeutic efficacy by injecting ACF and phosphate-buffered saline, as a control, into the dermis of photoaging nude mice and harvesting skin samples at weeks 1, 2, and 4 after treatment for assessment. The content of adipokines released from ACF was identified in vivo. The collagen synthesis and collagen degradation in ACF implants were evaluated by immune staining. Dermal thickness, fibroblast expression, collagen synthesis, ROS level, antioxidase expression, capillary density, and apoptotic cell number were evaluated by histological assessment, immune staining, and polymerase chain reaction in the skin samples. We demonstrated that ACF is the concentrated adipose extracellular matrix collagen fragment without viable cells and can be injected through fine needles. The lower expression of SA-β-gal, ROS and higher expression of antioxidase were observed in the ACF-treated group. ACF undergoes collagen degradation and promotes neocollagen synthesis in ACF implants. Meanwhile, ACF serves as a sustained-release system of adipokines and exhibits a significantly higher therapeutic effect on mouse skin photoaging by enhancing angiogenesis, antioxidant abilities, antiapoptotic activities, and collagen synthesis through sustainedly releasing adipokines. To sum up, ACF is an adipokines-enriched, sustained-release extracellular matrix collagen scaffold that can prevent UVA-induced skin photoaging in mice. ACF may serve as a novel autologous skin filler for skin rejuvenation applications in the clinic.

Adipose Collagen Fragment: an Injectable Sustained-release Collagen Scaffold of Adipokines for Preventing Skin Photoaging in Mice

Objective: Ultraviolet A (UVA) radiation is the major contributor to skin photoaging, associated with increased collagen degradation and reactive oxygen species expression. Adipokines have been proven as promising therapeutic agents for skin photoaging. However, adipokine therapy is generally limited by the short in vivo release duration and biological instability. Therefore, developing a treatment that provides a sustained release of adipokines and enhanced therapeutic effects is desirable. Methods: Adipose collagen fragment (ACF) was prepared from lipoaspirate and characterized. The injectability, collagen components, and adipokine release pattern of ACF were identified in vitro. Then, we evaluated its therapeutic efficacy by injecting ACF and phosphate-buffered saline, as a control, into the dermis of photoaging nude mice and harvesting skin samples at weeks 1, 2, and 4 after treatment for assessment. The collagen synthesis and collagen degradation in ACF implants were evaluated by immune staining. Dermal thickness, fibroblast expression, collagen synthesis, reactive oxygen species level, antioxidase expression, capillary density, and apoptotic cell number were evaluated by histological assessment, immune staining, and polymerase chain reaction in the skin samples.Results: ACF is the concentrated adipose extracellular matrix collagen fragment without viable cells and can be injected through fine needles. ACF undergoes collagen degradation and promotes neocollagen synthesis in ACF implants. Meanwhile, ACF serves as a sustained-release system of adipokines and exhibits a significantly higher therapeutic effect on mouse skin photoaging compared to controls. ACF increases the dermal thickness, improves fibroblast expression and promotes collagen synthesis. ACF treatment of UVA-irradiated skin reduces reactive oxygen species expression, decreases the number of apoptotic cells, improves capillary density and promotes the expression of antioxidase superoxide dismutase-1, catalase and glutathione peroxidase-1 by sustainedly releasing adipokines.Conclusions: ACF is an adipokines-enriched, sustained-release extracellular matrix collagen scaffold that can prevent UVA-induced skin photoaging in mice. ACF may serve as a novel autologous skin filler for skin rejuvenation applications in the clinic.

Recombinant Co-Expression of Collagen A1 (I) Fragment with the Prolyl 4-Hydroxylases (P4H) Subunits in Komagataella phaffii

Recombinant collagen and collagen-like products are increasingly replacing animal-sourced collagen that is difficult to produce in safe and standard quality. In this study to produce hydroxylated collagen, a 400 base pair collagen fragment of the bovine COL1A1 gene was co-expressed with prolyl-4-hydroxylase subunit α (P4Hα) and prolyl-4-hydroxylase subunit β(P4Hβ) encoding the P4H enzyme in Komagataella phaffii. For this purpose, each target gene was inserted into the pPICZαA vector and then cloned in E. coli DH5α cells. Subsequently, co-expression vectors were constructed using recombinant vectors isolated from positive clones according to the in vitro multimer ligation method. All recombinant expression and co-expression vectors were transformed into K. phaffii X33 cells by electroporation. The results of reverse transcriptase-polymerase chain reaction (PCR) proved that all target genes were transcribed by recombinant strains. The expression of recombinant proteins was performed for 96 hours by methanol-fed cultivation, and the concentration of the purified proteins from the culture medium was measured by the His-Tag enzyme-linked immunosorbent assay (ELISA) method. The concentrations of rP4Hα and rP4Hβ, and rCol1 proteins expressed individually by recombinant strains were determined to be 10.69 µg/L, 10.74 µg/L, and 8.61 µg/L, respectively, while the concentrations of co-expressed rP4Hα/β and rP4Hα/β/rCol1 proteins were 7.82 µg/L and 5.02 µg/L, respectively. These results showed that the target genes were successfully expressed and co-expressed in the recombinant K. phaffii cell.

Conjugates of Chitosan and Calcium Alginate with Oligoproline and Oligohydroxyproline Derivatives for Potential Use in Regenerative Medicine

New materials that are as similar as possible in terms of structure and biology to the extracellular matrix (external environment) of cells are of great interest for regenerative medicine. Oligoproline and oligohydroxyproline derivatives (peptides 2–5) are potential mimetics of collagen fragments. Peptides 2–5 have been shown to be similar to the model collagen fragment (H-Gly-Hyp-Pro-Ala-Hyp-Pro-OH, 1) in terms of both their spatial structure and biological activity. In this study, peptides 2–5 were covalently bound to nonwovens based on chitosan and calcium alginate. Incorporation of the peptides was confirmed by Fourier transform -infrared (FT-IR) and zeta potential measurements. Biological studies (cell metabolic activity by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and Live/Dead assay) proved that the obtained peptide-polysaccharide conjugates were not toxic to the endothelial cell line EA.hy 926. In many cases, the conjugates had a highly affirmative influence on cell proliferation. The results of this study show that conjugates of chitosan and calcium alginate with oligoproline and oligohydroxyproline derivatives have potential for use in regenerative medicine.

Collagen fragments quantified in serum as measures of desmoplasia associate with survival outcome in patients with advanced pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) patients have poor prognosis and poor response to treatment. This is largely due to PDAC being associated with a dense and active stroma and tumor fibrosis (desmoplasia). Desmoplasia is characterized by excessive degradation and formation of the extracellular matrix (ECM) generating collagen fragments that are released into circulation. We evaluated the association of specific collagen fragments measured in pre-treatment serum with outcome in patients with PDAC. Matrix metalloprotease (MMP)-degraded type I collagen (C1M), type III collagen (C3M), type IV collagen (C4M) and a pro-peptide of type III collagen (PRO-C3) were measured by ELISA in pre-treatment serum from a randomized phase 3 clinical trial of patients with stage III/IV PDAC treated with 5-fluorouracil based therapy (n = 176). The collagen fragments were evaluated for their correlation (r, Spearman) with serum CA19-9 and for their association with overall survival (OS) based on Cox-regression analyses. In this phase 3 PDAC trial, pre-treatment serum collagen fragment levels were above the reference range for 67%-98% of patients, with median values in PDAC approximately two-fold higher than reference levels. Collagen fragment levels did not correlate with CA19-9 (r = 0.049–0.141, p = ns). On a continuous basis, higher levels of all collagen fragments were associated with significantly shorter OS. When evaluating degradation (C3M) and formation (PRO-C3) of type III collagen further, higher PRO-C3 was associated with poor OS (>25th percentile cut-point, HR = 2.01, 95%CI = 1.33–3.05) and higher C3M/PRO-C3 ratio was associated with improved OS (>25th percentile cut-point, HR = 0.53, 95%CI = 0.34–0.80). When adjusting for CA19–9 and clinical covariates, PRO-C3 remained significant (HR = 1.65, 95%CI = 1.09–2.48). In conclusion, collagen remodeling quantified in pre-treatment serum as a surrogate measure of desmoplasia was significantly associated with OS in a phase 3 clinical PDAC trial, supporting the link between desmoplasia, tumorigenesis, and response to treatment. If validated, these biomarkers may have prognostic and/or predictive potential in future PDAC trials.