Effect of prophylactic treatment of polyphenols on collagen-induced arthritis (CIA).

<p>(A) Hematoxylin and Eosin-stained sections and (B) Masson's Trichrome stained sections of tibiofemoral joints of rats; NC indicates Negative Control (i.e., normal joint without induction and treatment); remaining illustrations are from CIA joints: PPC—Prophylactic Positive Control; PE—Prophylactic EGCG; and PT—Prophylactic TA; (black arrow = disintegrated cartilage; green arrows = intact cartilage)</p

Paw volume and cartilage degradation scoring of prophylactically treated groups.

<p>(A) Paw volume changes in prophylactically treated groups from day -18 to 43 (values analyzed using RM- ANOVA with Graph Pad prism); "***" indicates significant difference compared to NC (P<0.001); "#" indicates significant difference compared to PPC (### represents P<0.001 and # represents P<0.05); values represented in Mean ± SEM, n = 12. (B) Cartilage degradation scores from histomorphological sections of prophylactically treated groups; "**" indicates significant difference from NC (P<0.01); statistically significant differences between PPC and PT or PE groups indicated as "#" (P<0.05); values represented in Mean ± SEM, n = 6.</p

Percentage of collagen degradation and release of GAGs from bovine AC (with and without polyphenols) treated with collagenase.

<p>^aData were analyzed using one-way ANOVA with Bonferroni post hoc test. The EGCG (P<0.01), TA (P<0.05), and CAT (P<0.05) treated groups showed significantly reduced percent degradation of collagen compared to controls. Similarly, the percent release of GAG in the EGCG- (P<0.01) and TA-treated (P<0.01) groups had significantly reduced (P-value: 0.0268) compared to controls but no statistical significance was observed in CAT and QUE. The values are represented as Mean ± SEM, n = 3.</p><p>(* indicates significant difference in comparison to control, P<0.05.)</p><p>Percentage of collagen degradation and release of GAGs from bovine AC (with and without polyphenols) treated with collagenase.</p

Intra-Articular Injections of Polyphenols Protect Articular Cartilage from Inflammation-Induced Degradation: Suggesting a Potential Role in Cartilage Therapeutics

<div><p>Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, inflict an enormous health care burden on society. Osteoarthritis, a degenerative joint disease with high prevalence among older people, and rheumatoid arthritis, an autoimmune inflammatory disease, both lead to irreversible structural and functional damage to articular cartilage. The aim of this study was to investigate the effect of polyphenols such as catechin, quercetin, epigallocatechin gallate, and tannic acid, on crosslinking type II collagen and the roles of these agents in managing <i>in vivo</i> articular cartilage degradation. The thermal, enzymatic, and physical stability of bovine articular cartilage explants following polyphenolic treatment were assessed for efficiency. Epigallocatechin gallate and tannic acid-treated explants showed >12 °C increase over native cartilage in thermal stability, thereby confirming cartilage crosslinking. Polyphenol-treated cartilage also showed a significant reduction in the percentage of collagen degradation and the release of glycosaminoglycans against collagenase digestion, indicating the increase physical integrity and resistance of polyphenol crosslinked cartilage to enzymatic digestion. To examine the <i>in vivo</i> cartilage protective effects, polyphenols were injected intra-articularly before (prophylactic) and after (therapeutic) the induction of collagen-induced arthritis in rats. The hind paw volume and histomorphological scoring was done for cartilage damage. The intra-articular injection of epigallocatechin gallate and tannic acid did not significantly influence the time of onset or the intensity of joint inflammation. However, histomorphological scoring of the articular cartilage showed a significant reduction in cartilage degradation in prophylactic- and therapeutic-groups, indicating that intra-articular injections of polyphenols bind to articular cartilage and making it resistant to degradation despite ongoing inflammation. These studies establish the value of intra-articular injections of polyphenol in stabilization of cartilage collagen against degradation and indicate the unique beneficial role of injectable polyphenols in protecting the cartilage in arthritic conditions.</p></div

Compression analysis of AC.

<p>Compressive load at 50% compression of AC incubated 4 days without and with polyphenols 200-μM (CAT, QUE, EGCG, TA) prepared using PBS; figure shows day-4 results; no significant difference (P>0.05) was observed between the control and polyphenol-treated samples; values are represented in Mean ± SEM, n = 3.</p

Increase in thermal denaturation temperature of polyphenol-treated cartilage with reference to native untreated bovine cartilage determined using DSC.

<p>^a n = 2; experiments were performed on two independent sets of bovine AC.</p><p>Increase in thermal denaturation temperature of polyphenol-treated cartilage with reference to native untreated bovine cartilage determined using DSC.</p

Differential scanning calorimetric (DSC) analysis.

<p>Thermograms of control (native) and polyphenol-treated (CAT, QUE, EGCG, and TA) bovine AC samples (representative picture)</p

Paw volume and cartilage degradation scoring of therapeutically treated groups.

<p>(A) Paw volume changes in therapeutically treated rat groups from day 1–43 (values analyzed using RM-ANOVA with Graph Pad prism; "***" indicates significant difference compared to negative control (NC) (P<0.001); "#" indicates significant difference compared to therapeutic positive control (TPC) (### represents P<0.001, ## represents P<0.01 and # represents P<0.05); values represented in Mean ± SEM, n = 12. (B) Cartilage degradation scores from histomorphological sections of therapeutically treated groups; "**" indicates significant difference compared to NC (P<0.01); statistically significant differences between TPC and TT or TE groups indicated as "#" (P<0.05); values represented in Mean ± SEM, n = 6.</p

Sol–Gel Assisted Fabrication of Collagen Hydrolysate Composite Scaffold: A Novel Therapeutic Alternative to the Traditional Collagen Scaffold

Collagen is one of the most widely used biomaterial for various biomedical applications. In this Research Article, we present a novel approach of using collagen hydrolysate, smaller fragments of collagen, as an alternative to traditionally used collagen scaffold. Collagen hydrolysate composite scaffold (CHCS) was fabricated with sol–gel transition procedure using tetraethoxysilane as the silica precursor. CHCS exhibits porous morphology with pore sizes varying between 380 and 780 μm. Incorporation of silica conferred CHCS with controlled biodegradation and better water uptake capacity. Notably, 3T3 fibroblast proliferation was seen to be significantly better under CHCS treatment when compared to treatment with collagen scaffold. Additionally, CHCS showed excellent antimicrobial activity against the wound pathogens <i>Staphylococcus aureus, Bacillus subtilis</i>, and <i>Escherichia coli</i> due to the inherited antimicrobial activity of collagen hydrolysate. In vivo wound healing experiments with full thickness excision wounds in rat model demonstrated that wounds treated with CHCS showed accelerated healing when compared to wounds treated with collagen scaffold. These findings indicate that the CHCS scaffold from collagen fragments would be an effective and affordable alternative to the traditionally used collagen structural biomaterials

Molecular Level Insights on Collagen–Polyphenols Interaction Using Spin–Relaxation and Saturation Transfer Difference NMR

Interaction of small molecules with collagen has far reaching consequences in biological and industrial processes. The interaction between collagen and selected polyphenols, viz., gallic acid (GA), pyrogallol (PG), catechin (CA), and epigallocatechin gallate (EGCG), has been investigated by various solution NMR measurements, viz., ¹H and ¹³C chemical shifts (δ_H and δ_C), ¹H nonselective spin–lattice relaxation times (<i>T</i>_1NS) and selective spin–lattice relaxation times (<i>T</i>_1SEL), as well as spin–spin relaxation times (<i>T</i>₂). Furthermore, we have employed saturation transfer difference (STD) NMR method to monitor the site of GA, CA, PG, and EGCG which are in close proximity to collagen. It is found that −COOH group of GA provides an important contribution for the interaction of GA with collagen, as evidenced from ¹³C analysis, while PG, which is devoid of −COOH group in comparison to GA, does not show any significant interaction with collagen. STD NMR data indicates that the resonances of A-ring (H2′, H5′ and H6′) and C-ring (H6 and H8) protons of CA, and A-ring (H2′ and H6′), C-ring (H6 and H8), and D-ring (H2″and H6″) protons of EGCG persist in the spectra, demonstrating that these protons are in spatial proximity to collagen, which is further validated by independent proton spin-relaxation measurement and analysis. The selective ¹H <i>T</i>₁ measurements of polyphenols in the presence of protein at various concentrations have enabled us to determine their binding affinities with collagen. EGCG exhibits high binding affinity with collagen followed by CA, GA, and PG. Further, NMR results propose that presence of gallic acid moiety in a small molecule increases its affinity with collagen. Our experimental findings provide molecular insights on the binding of collagen and plant polyphenols