Electrospun Scaffold Micro-Architecture Induces an Activated Transcriptional Phenotype within Tendon Fibroblasts

Biomaterial augmentation of surgically repaired rotator cuff tendon tears aims to improve the high failure rates (∼40%) of traditional repairs. Biomaterials that can alter cellular phenotypes through the provision of microscale topographical cues are now under development. We aimed to systematically evaluate the effect of topographic architecture on the cellular phenotype of fibroblasts from healthy and diseased tendons. Electrospun polydioxanone scaffolds with fiber diameters ranging from 300 to 4000 nm, in either a highly aligned or random configuration, were produced. Healthy tendon fibroblasts cultured for 7 days on scaffolds with highly aligned fibers demonstrated a distinctive elongated morphology, whilst those cultured on randomly configured fibers demonstrated a flattened and spread morphology. The effect of scaffold micro-architecture on the transcriptome of both healthy and diseased tendon fibroblasts was assessed with bulk RNA-seq. Both healthy (n = 3) and diseased tendon cells (n = 3) demonstrated a similar transcriptional response to architectural variants. Gene set enrichment analysis revealed that large diameter (≥2000 nm) aligned scaffolds induced an upregulation of genes involved in cellular replication and a downregulation of genes defining inflammatory responses and cell adhesion. Similarly, PDPN and CD248, markers of inflammatory or “activated” fibroblasts, were downregulated during culture of both healthy and diseased fibroblasts on aligned scaffolds with large (≥2000 nm) fiber diameters. In conclusion scaffold architectures resembling that of disordered type III collagen, typically present during the earlier phases of wound healing, resulted in tendon fibroblast activation. Conversely, scaffolds mimicking aligned diameter collagen I fibrils, present during tissue remodelling, did not activate tendon derived fibroblasts. This has implications for the design of scaffolds used during rotator cuff repair augmentation.

Effect of Chitosan Deacetylation on Its Affinity to Type III Collagen: A Molecular Dynamics Study

The ability to form strong intermolecular interactions by linear glucosamine polysaccharides with collagen is strictly related to their nonlinear dynamic behavior and hence bio-lubricating features. Type III collagen plays a crucial role in tissue regeneration, and its presence in the articular cartilage affects its bio-technical features. In this study, the molecular dynamics methodology was applied to evaluate the effect of deacetylation degree on the chitosan affinity to type III collagen. The computational procedure employed docking and geometry optimizations of different chitosan structures characterized by randomly distributed deacetylated groups. The eight different degrees of deacetylation from 12.5% to 100% were taken into account. We found an increasing linear trend (R2 = 0.97) between deacetylation degree and the collagen–chitosan interaction energy. This can be explained by replacing weak hydrophobic contacts with more stable hydrogen bonds involving amino groups in N-deacetylated chitosan moieties. In this study, the properties of chitosan were compared with hyaluronic acid, which is a natural component of synovial fluid and cartilage. As we found, when the degree of deacetylation of chitosan was greater than 0.4, it exhibited a higher affinity for collagen than in the case of hyaluronic acid.

Cosmetic Potential of a Recombinant 50 kDa Protein

Collagen and its derivative proteins have been widely used as a major component for cosmetic formulations as a natural ingredient and moisturizer. Most commercially available collagens are animal-derived collagen type I and other forms of collagen, such as type III collagen, are far less prevalent in animals, making extraction and purification extremely difficult and expensive. Here, we report the production of a 50 kDa protein produced in yeast that is 100% identical to the N-terminus of the human type III collagen. This recombinant protein has a larger molecular weight than most incumbent recombinant collagen proteins available for personal care applications. We report the industrialization of both the fermentation and purification processes to produce a final recombinant protein product. This final protein product was shown to be safe for general applications to human skin and compatible with common formulation protocols, including ethanol-based formulations. This recombinant collagen type III protein was also shown to uniquely stimulate both collagen type I and type III production and secretion by primary human dermal fibroblasts. The unique combination of biostimulation, compatibility with beauty product formulations and demonstrated commercial production, make this novel recombinant type III collagen a good candidate for broad application in the cosmetics industry.

Double Layer Socket Preservation Technique Associated with Xenogenous Bone Graft and Polypropylene Membrane: A Case Report

With the demand for tooth/gum aesthetics in implant-supported rehabilitations, the surgeon, whether an implant specialist or not, increasingly needs to be mindful of proper care for socket preservation following extraction. The paper presented here reports the case of a male patient who manifested dental impairment of the Upper Left First Molar (tooth #26) [in FDI notation]; following tomographic analysis and after reaching a consensus with the patient, the decision was made to extract said tooth and preserve the socket for subsequent implant placement. The aim of the case report is to present a clinical case of alveolar ridge preservation through the “Double Layer Socket Preservation” technique, a technique created by Barry Barthee, whereby a xenogenous graft under an xenogenous type III collagen membrane was combined with a polypropylene barrier. Following research and study results on the subject, it was concluded that by applying this technique, the alveolar ridge is greatly preserved and bone volume is maintained, both of which are very important factors for good health of the tissues surrounding the implant and consequent increase in the survival of the implant itself.

Synergistic collagen-condiment: Streptococcal collagen-like (Scl) protein in cell-adhesion and diabetic wound-closure matrix

Group A streptococcus (GAS), Streptococcus pyogenes manifests plethora of diseases through its explicit virulence factors. Among these, the recently deciphered MSCRAMMs, Streptococcal collagen-like (Scls) adhesins are most studied proteins in context of their biophysically stable collagenous-sequence (Gly-X-Y) despite the difference from analogous mammalian-collagen. Based on recent evidence on collagen-mimetic Scls, we elucidated biomaterial-potential of the unmodified, recombinant Scl1 (rScl1). Initially, rScl1 trimeric- assembly yielded its stability in silico than the monomeric-unit. Thereby, rScl1 matrix characterization was confirmed in vitro. rScl1 exhibited high A549 and HepG2 cell- viability—rScl1 dose incremented to 20.0 µg/ml at time points up to 24 hr, and on 24 hr stored-dishes—deliberating it non-cytotoxic. Imploring cell-adhesion potential, we observed increased cell-counts tangential to rScl1-gradient. This affirmative prelude on rScl1 as a supporting-matrix cued its synergy to collagen; we discerned it through rScl1-augmented, full-thickness diabetic wound-closure in vivo and as a first, we studied > 18-month rabbit alloxan-models. We have ascertained re-epithelialization with higher type III collagen in absence of inflammation evidenced morphometrically and histologically. Finally, we correlated our observations through atomistic-evaluation of rScl1-α2β1-integrin interaction, surprisingly, with augmented binding-energy compared to collagen. Hence, connoting recombinant-streptococcal collagen as an ‘alternate’; with further characterization, rScl1 can potentiate important revelations conceding homogeneous and safe, bio-available, biomaterial.

Jiedu Tongluo Decoction Attenuates Myocardial Fibrosis Through Inhibition of The TGF-β1/Smad2/3 Pathway

Background: Jiedu Tongluo (JDTL) decoction is a traditional Chinese medicine (TCM) formula containing three herbal ingredients that is widely used to treat myocardial fibrosis (MF). This study aimed to investigate the molecular mechanism of action of JDTL decoction for the treatment of MF. Methods: The chemical constituents of Traditional Chinese medicine were analyzed by HPLC, Forty Wistar rats were randomly divided into normal control group, model group, Jiedu Tongluo decoction low-dose and high-dose treatment groups, with 10 rats in each group. Rat myocardial fibrosis model was induced by subcutaneous injection of 5 mg•kg-1 isoproterenol hydrochloride. 5 and 50 g•(kg.d) -1 were given intragastric administration for 7 days. Hematoxylin-eosin (HE) and Masson staining were performed for histological evaluation of myocardial tissue, MTT method detects the activity of cardiac fibroblasts, The alkaline water method is used to determine the content of hydroxyproline in myocardial tissue and myocardial fibroblasts, Immunohistochemical method was used to detect the expression of myocardial tissue transforming growth factor (TGF) β1, p smad2/3, and type III collagen (Col III) expression, Immunofluorescence to detect the expression of α-SMA/Vimentin in myocardial tissue and myocardial fibroblasts; Western blot was used to detect the protein expression levels of TGFβ1 and psmad2/3 in cardiac fibroblasts.Results: In this study, six compounds of JDTL decoction were identified by high-performance liquid chromatography (HPLC). Hematoxylin and eosin (HE) and Masson staining showed that in the isoproterenol hydrochloride (ISO)-induced MF rat model, JDTL treatment protected the myocardial structure and inhibited type III collagen (COL3) expression(p<0.05). The immunohistochemistry (IHC) results also showed that JDTL treatment significantly reduced the expression of vimentin, alpha-smooth muscle actin (α-SMA), and transforming growth factor-beta 1 (TGF-β1) as well as the phosphorylation of Smad2/3 in the rat MF model(p<0.05). Rat cardiac fibroblasts (RCFs) were used for the following assays performed in vitro: hydroxyproline detection, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound-healing test, western blot, and double immunofluorescence staining; the results of these assays confirmed the inhibitory effects of JDTL decoction on the proliferation, migration, and transdifferentiation abilities of RCFs as well as the molecular mechanisms underlying these effects, including the inhibition of the TGF-β1/Smad2/3 pathway through downregulation of TGF-β1 expression and phosphorylation of Smad2/3 as well as inhibition of vimentin and α-SMA expression(p<0.05). Conclusion: JDTL decoction can prolong the process of MF through inhibition of the TGF-β1/Smad2/3 signaling pathway.

22 The potential of serum autoantibodies against type III collagen in cancer

BackgroundAutoantibodies are classically associated with autoimmune diseases but have recently emerged as attractive cancer biomarkers as they can be easily assessed in serum. Certain autoantibodies have been shown to promote cancer while others contribute to the body’s defense against it. Cancer progression is associated with excessive remodeling of the extracellular matrix (ECM) and collagen, but little is known about the role of autoantibodies against collagen in cancer. To investigate autoreactivity against collagen in cancer, we developed a novel biomarker assay to quantify autoantibodies against type III collagen products in serum from patients with various solid tumor types and compared levels with those find in healthy controls.MethodsThe presence and levels of autoantibodies against denatured type III collagen were measured in pretreatment serum from 223 patients with bladder cancer (n=20), breast cancer (n=20), colorectal cancer (n=20), head and neck cancer (n=20), kidney cancer (n=20), liver cancer (n=3), lung cancer (n=20), melanoma (n=20), ovarian cancer (n=20), pancreatic cancer (n=20), prostate cancer (n=20), and stomach cancer (n=20), and compared to age-matched healthy controls (n=33). Statistical differences were analyzed using the Kruskal-Wallis test adjusted for Dunn’s multiple comparisons test.ResultsSerum levels of autoantibodies against type III collagen were significantly lower in patients with bladder cancer (p=0.0007), breast cancer (p=0.0002), colorectal cancer (p<0.0001), head and neck cancer (p=0.0005), kidney cancer (p=0.005), liver cancer (p=0.030), lung cancer (p=0.0004), melanoma (p<0.0001), ovarian cancer (p<0.0001), pancreatic cancer (p<0.0001), prostate cancer (p<0.0001), and stomach cancer (p<0.0001) compared to healthy controls. This autoimmunity biomarker could discriminate between cancer and healthy controls with an AUROC value of 0.88 (p<0.0001).ConclusionsIn this study, we observed that cancer patients with different solid tumor types have downregulated levels of circulating autoantibodies directed against type III collagen compared to healthy controls suggesting that autoantibodies against type III collagen and tumor fibrosis may be important for tumor control and eradication. This autoimmunity biomarker may have the potential for studying and monitoring the close relationship between autoimmunity and cancer such as the risk of developing cancer on rheumatoid arthritis immunosuppressant or the risk of developing immune-related adverse events on cancer immunotherapy.Ethics ApprovalThe serum samples in this study were obtained from the commercial vendor Proteogenex and BioIVT, and according to the vendors, sample collection was approved by an Institutional Review Board or Independent Ethical Committee and patients gave their informed consent (Protocol numbers PG-ONC 2003/1 and WIRB® Protocol #20161665). All investigations were carried out according to the Helsinki Declaration.

Physical Training as a Blood Pressure Reducer and a Remodeler of Cardiac Fibers in Spontaneously Hypertensive Rats (SHR)

Background: Hypertension is the most prevalent of all cardiovascular diseases, reaching target organs such as the heart. Blood pressure control is critical for preventing organ damage induced by hypertension. Objective: To analyze blood pressure, heart rate, left ventricular thickness, the percentage of cardiac fibrosis and the percentage of type III collagen in Spontaneously Hypertensive Rats (SHR) submitted to swimming physical training. Methods: The experimental groups were composed of male Wistar Kyoto (WKY) rats (309-311g), which were divided into: 1) Normotensive Sedentary group (SN) (n = 6); 2) Trained Normotensive group (TN) (n = 6); 3) Sedentary Hypertensive group (SH) (n = 6); 4) Trained Hypertensive group (TH) (n = 6). After the end of the protocol, the animals were initially anesthetized to measure blood pressure. Results: Physical training was responsible for decreasing blood pressure (F = 16,968; p <0.001) and heart rate (F = 10.710; p = 0.004) in the trained groups (normotensive and hypertensive). Moreover, training was responsible for providing an increase in the thickness of the left ventricle (F = 7,254; p = 0.014) and a reduction in the percentage of cardiac fibrosis (F = 16,081; p <0.001). Furthermore, it was observed that the trained group had lower values of type III collagen (F = 13,166; p = 0.002). Conclusions: Physical swimming training triggered a decrease in blood pressure, heart rate, the percentage of fibrosis and the percentage of type III collagen. In addition, there was also a cardiac remodeling due to the increase in left ventricular hypertrophy.

Copaiba oil-resin (Copaifera langsdorfii Desf.: Caesalpiniaceae) associated with laser therapy for skin wound treatment in Wistar rats

Alternative protocols for the treatment of skin lesions have been developed with the use of techniques such as photobiomodulation and phytotherapy, aiming to optimize this process. To evaluate the effectiveness of copaiba (Copaiferalangsdorffii) oil-resin and low-level laser therapy for treating cutaneous wounds, 15 Wistar rats (Rattusnorvergicus) were used, in whom five 8-mm lesions were produced. The following protocols were applied: negative control group (T1); positive control group (T2); laser therapy with AsGa (904 nm), continuous, focal mode for 10 s, dosage of 4 J/cm² (T3); copaiba oil-resin (T4); and association group (copaiba and low-level laser) (T5). The efficacy of each technique was evaluated based on macroscopic aspects of the lesion, wound healing rate, and histopathological analysis (inflammatory infiltrate and collagen expression). The Kruskal-Wallis test was used for statistical analyses (P> 0.05). Copaiba treatment showed an advantage in type III collagen expression, whereas laser therapy demonstrated an enhanced capacity for tissue regeneration. The significant advantage obtained from the association treatment is the improvement of the macroscopic aspect of the wound, with a reduction in crust formation.