Hyoepiglottic ligament collagen and elastin fiber composition and changes associated with aging.

OBJECTIVE: The epiglottis may contribute to upper airway obstruction in approximately 10% of patients with obstructive sleep apnea. Clinical experience indicates that older patients may be more likely to have epiglottis-related obstruction. This study was designed to examine tissue characteristics of the hyoepiglottic ligament as a possible factor in epiglottis-related obstruction based on previous research suggesting that older adults have fewer collagen, elastin, and muscle fibers in the hyoepiglottic ligament. METHODS: This is a cross-sectional study of 25 human cadaver hyoepiglottic ligaments. Specimens were stained using Masson's trichrome and Picrosirius red for collagen fibers and with Verhoeff-Van Gieson for elastin fibers. Percentage of collagen and elastin fiber staining for each specimen was calculated and averaged over three regions of each ligament section. Regression analysis was used to determine the association between age, smoking history, and collagen and elastin composition of the hyoepiglottic ligament. RESULTS: The average age of the specimens was 68.4 ± 15.1 years (range 30-90 years). Increasing age was associated with a lower percentage of collagen and elastin fibers. When accounting for tobacco use, each 1-year increase in age was associated with a 0.53% decrease in Masson's trichrome staining (P = 0.004), a 0.35% decrease in Picrosirius red staining (P = 0.023), and a 0.33% decrease in Verhoeff-Van Gieson staining (P = 0.008). CONCLUSION: Increasing age is associated with decreases in the collagen and elastin content of the hyoepiglottic ligament. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:1245-1248, 2018.

The synergetic effect of hydrogel stiffness and growth factor on osteogenic differentiation.

Cells respond to various chemical signals as well as environmental aspects of the extracellular matrix (ECM) that may alter cellular structures and functions. Hence, better understanding of the mechanical stimuli of the matrix is essential for creating an adjuvant material that mimics the physiological environment to support cell growth and differentiation, and control the release of the growth factor. In this study, we utilized the property of transglutaminase cross-linked gelatin (TG-Gel), where modification of the mechanical properties of TG-Gel can be easily achieved by tuning the concentration of gelatin. Modifying one or more of the material parameters will result in changes of the cellular responses, including different phenotype-specific gene expressions and functional differentiations. In this study, stiffer TG-Gels itself facilitated focal contact formation and osteogenic differentiation while soft TG-Gel promoted cell proliferation. We also evaluated the interactions between a stimulating factor (i.e. BMP-2) and matrix rigidity on osteogenesis both in vitro and in vivo. The results presented in this study suggest that the interactions of chemical and physical factors in ECM scaffolds may work synergistically to enhance bone regeneration.

Injectable gel graft for bone defect repair.

AIM: To examine the performance of an injectable gel graft made of transglutaminase (Tg)-crosslinked gelatin gel with BMP-2 (BMP-2-Tg-Gel) for bone defect repair in animal models. MATERIALS & METHODS: BMP-2 mixed with gelatin gel was crosslinked using Tg. The release of tethered BMP-2 through autocrine and paracrine pathways was demonstrated by using C2C12 and NIH 3T3 cells, respectively. BMP-2-Tg-Gel was injected into the induced cranial defect site. After 14 days, the sample was removed for x-ray imaging and histological evaluation. RESULTS: Our in vivo results demonstrated that the injectable Tg-Gel with its osteoconductivity and controllable BMP-2 activity induced bone formation in our rat models when tethered with BMP-2. CONCLUSION: Tg-Gel as an injectable functional bone graft may enable the use of minimally invasive surgical procedures to treat irregular-shaped bone defects. Furthermore, this novel approach is capable of incorporating and controlling the release of therapeutic agents that may advance the science of tissue regeneration.

Delivery of demineralized bone matrix powder using a thermogelling chitosan carrier.

Demineralized bone matrix (DBM) powder is widely used for bone regeneration due to its osteoinductivity and osteoconductivity. However, difficulties with handling, its tendency to migrate from graft sites, and lack of stability after surgery can sometimes limit the clinical utility of this material. In this work, the possibility of using a thermogelling chitosan carrier to deliver DBM powder was assessed. The DBM-thermogelling putty improved handling and formed a gel-like composite in situ at body temperature within a clinically relevant time period. The properties of the formed composite, including morphology, porosity, mechanical properties, equilibrium swelling as well as degradability, are significantly influenced by the ratio of DBM to thermogelling chitosan. The in vitro study showed that the alkaline phosphatase activity of C2C12 cells encapsulated in the composite was steadily increased with culture time. The in vivo study showed that increased DBM content in the DBM-thermogelling chitosan induced ectopic bone formation in a nude rat model. The diffusion of growth factor from the DBM-thermogelling chitosan as well as the host-implant interactions are discussed.

Cell delivery using an injectable and adhesive transglutaminase-gelatin gel.

In this study, we developed an injectable gelatin-transglutaminase (TGase) gel for cell delivery. The procedure provides a minimally invasive approach to deliver cells into tissue in a manner that improves localization. The results indicate gelatin-TGase to be noncytotoxic and to have adhesive properties that help localize and prevent the scattering of the cells after delivery. The in situ crosslinking between gelatin chains and endogenous collagen can create a strong attachment between the gel and tissue extracellular matrix, preventing cells from dissipation. The gelatin-TGase was also shown to maintain the carried cells to be viable and proliferative. Finally, through the adjustment of the enzymatic crosslinker concentration, the release rate of the cells into the surrounding tissue after injection was demonstrated to be controllable.

Intra-articular injection of collagenase induced experimental osteoarthritis of the lumbar facet joint in rats.

We aimed to establish an animal model to investigate primary osteoarthritis of the lumbar facet joints after collagenase injection in rats and its effects on chondrocyte apoptosis. We hypothesized that osteoarthritic-like changes would be induced by collagenase injection and that apoptosis of chondrocytes would increase. Collagenase (1, 10, or 50 U) or saline (control) was injected into the lumbar facet joints. The histology and histochemistry of cartilage, synovium, and subchondral bone were examined at 1, 3, and 6 weeks after surgery. Apoptotic cells induced by 1 U of collagenase were quantified using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Degeneration of the cartilage and changes to the synovium and subchondral bone were dependent on both the doses of collagenase and the time after surgery. There were significantly more apoptotic chondrocytes in collagenase-treated joints than in control (P < 0.001 at 1 and 3 weeks and P < 0.05 at 6 weeks). Thus, lumbar facet joints subjected to collagenase developed osteoarthritic-like changes that could be quantified and compared. This model provides a useful tool for further study on the effects of compounds that have the potential to inhibit enzyme-associated damage to cartilage.

Are we getting enough sulfur in our diet?

Sulfur, after calcium and phosphorus, is the most abundant mineral element found in our body. It is available to us in our diets, derived almost exclusively from proteins, and yet only 2 of the 20 amino acids normally present in proteins contains sulfur. One of these amino acids, methionine, cannot be synthesized by our bodies and therefore has to be supplied by the diet. Cysteine, another sulfur containing amino acid, and a large number of key metabolic intermediates essential for life, are synthesized by us, but the process requires a steady supply of sulfur.Proteins contain between 3 and 6% of sulfur amino acids. A very small percentage of sulfur comes in the form of inorganic sulfates and other forms of organic sulfur present in foods such as garlic, onion, broccoli, etc.The minimal requirements (RDA) for all the essential amino acids have always been estimated in terms of their ability to maintain a nitrogen balance. This method asses amino acid requirements for protein synthesis, only one of the pathways that methionine follows after ingestion. To adequately evaluate the RDA for methionine, one should perform, together with a nitrogen balance a sulfur balance, something never done, neither in humans nor animals.With this in mind we decided to evaluate the dietary intake of sulfur (as sulfur amino acids) in a random population and perform sulfur balance studies in a limited number of human volunteers. Initially this was done to try and gain some information on the possible mode of action of a variety of sulfur containing compounds (chondroitin sulfate, glucosamine sulfate, and others, ) used as dietary supplements to treat diseases of the joints. Out of this study came information that suggested that a significant proportion of the population that included disproportionally the aged, may not be receiving sufficient sulfur and that these dietary supplements, were very likely exhibiting their pharmacological actions by supplying inorganic sulfur.

Chitosan/gelatin-based films crosslinked by proanthocyanidin.

Novel polymer networks consisting of crosslinked gelatin/chitosan were prepared by a solution casting technique. Methods for bulk crosslinking were developed to modify the gelatin/chitosan blends with the use of a nontoxic crosslinking reagent, proanthocyanidin (PA). FTIR spectral analyses of the preparations showed network formations of crosslinked gelatin, chitosan, and PA by amide and ester linkages. The crosslinked networks were stable in the aqueous state, and had improved mechanical properties and thermal stability when compared with nonlinked gelatin (G) and chitosan/gelatin (C/G) films. In vitro protease digestion and cell-culture studies showed that the PA-crosslinked C/G films are nontoxic and exhibited decreased biodegradation rate and a better ability to support cell adhesion and proliferation than noncrosslinked gelatin or chitosan alone. These results suggest that such a nontoxic crosslinked gelatin/chitosan scaffold can become a promising matrix for tissue-engineering applications.

Transdermal delivery of amino acids and antioxidants enhance collagen synthesis: in vivo and in vitro studies.

One of the most visible changes associated with the aging process in humans relates to a progressive thinning of the skin. This results from a decline in both collagen and glycosaminoglycans, as well as from changes in their chemical structure and 3-dimentional organization. Transdermal administration of antioxidants, a -lipoic acid (LA) (0.5%) and proanthocyanidin PA) (0.3%) in a standard cosmetic vehicle base formulation supplemented with 2% benzyl alcohol as a penetration enhancer, a mixture of essential amino acids (0.2%), significantly enhanced collagen synthesis and deposition. The amino acid mixture was designed to mimic serum concentrations, with supplemental methionine added to provide additional sulfur. The histological appearance of the skin of mature female rats treated in this fashion reflected the increased deposition of collagen in the dermis as well as a thickened epidermal layer. The changes do not seem to be mediated by TGF- ss or PDGF, two growth factors known to stimulate collagen synthesis. At lower concentrations, a -lipoic acid did not affect cell proliferation but at higher doses, while it had an inhibitory effect on (3)H-thimidine uptake, it did enhance collagen production. Pronanthocyanidin did not affect cell proliferation but significantly increased collagen synthesis by cultured fibroblasts.

Combined effects of phosphatidylcholine and demineralized bone matrix on bone induction.

The osteoinductivity of demineralized bone matrix (DBM) becomes significantly reduced if the specimens are further delipidated with chloroform-methanol. The addition of phosphatidylcholine (PC), a major constituent of the lipid fraction present in the calcification front during normal bone formation, can restore the biological activity. Active endochondral bone formation is observed in the DBM/PC implants placed in the anterior abdominal wall musculature or subcutaneously for 28 days. When PC is added to generate a putty containing 60% PC and 40% DBM, biochemical parameters associated with osteoinductivity are significantly enhanced. Biological responses evaluated histologically and by determination of alkaline phosphatase activity are in very good agreement. The DBM/PC putty has good handling properties, can be molded into different shapes, and does not wash away from the application site. An advantage of adding PC is that it not only enhances the handling properties, but also boosts the osteoinductivity of the preparation.

Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix.

A sensitive, rapid, reliable and quantitative method to check the bone forming potential of demineralized bone matrix (DBM) has been developed. The osteoinductivity of the bone morphogenetic proteins (BMPs), present in DBM, can be measured in vitro using a pluripotent myoblast C2C12 cell line. Alkaline phosphatase activity induced by co-incubation of DBM with C2C12 cells was dose-responsive and corresponds to the amount of active BMPs in DBM. Bone forming potential was simultaneously tested in vivo by implanting DBM intra-muscularly in nude rats. ALP activity induced in C2C12 cells, correlated with bone formation in vivo (r=0.88), determined by alkaline phosphatase activity, mineralization density and histomorphology of the DBM explants. Results from DBM batches, originating from five established Bone Banks, showed good consistency between in vitro and in vivo assays. However, DBM activity varied widely from bank to bank as well as from batch to batch within the same bank.

Proanthocyanidin: a natural crosslinking reagent for stabilizing collagen matrices.

While attempting to find a suitable crosslinking reagent for biopolymers, a naturally occurring proanthocyanidin (PA) obtained from grape seeds was selected to fix biological tissues. The cytotoxicity and crosslinking rate, reflected by the in vitro and in vivo degradation of fixed matrices has been studied. The shrinkage temperature of the fixed bovine pericardium increased from 66 to 86 degrees C. A cytotoxicity assay using fibroblast cultures revealed that PA is approximately 120 times less toxic than glutaraldehyde (GA), a currently used tissue stabilizer. In vitro degradation studies showed that fixed tissue was resistant to digestion by bacterial collagenase. Crosslinks between PA and tissues can be stabilized by decreasing the dielectric constant of the solution during storage. After subcutaneous implantation for periods ranging between 3 and 6 weeks, we found no apparent degradation of the GA- or PA-fixed tissues, whereas fresh tissue controls rapidly disintegrated. Beyond 6 weeks PA crosslinks began to degrade. More fibroblasts migrated and proliferated inside the PA-fixed implants compared with GA counterparts. Tissues crosslinked with PA manifested an enhanced collagen expression and deposition and did not calcify after implantation. GA, on the other hand, even after thorough rinsing continued to be cytotoxic, inhibited collagen synthesis and encouraged dystrophic calcification. Collagen matrices crosslinked with PA are expected to be of value in the design of matrices that will encourage cell ingrowth and proliferation, which are temporary in nature, and that are intended to regenerate or replace missing tissues, which can delay the biogradation of collagen. As such they should be of significant value in the emerging field of tissue engineering.

Collagen-targeted BMP3 fusion proteins arrayed on collagen matrices or porous ceramics impregnated with Type I collagen enhance osteogenesis in a rat cranial defect model.

Bone morphogenetic protein 3 (BMP3) is a potent osteoinductive growth factor belonging to the TGF-beta superfamily. In this study, we engineered a recombinant BMP3 protein to include an auxiliary collagen-targeting domain derived from von Willebrand coagulation factor (vWF). The collagen-targeted BMP3 fusion protein (rhBMP3-C) was expressed in E. coli, purified from bacterial inclusion bodies, renatured under controlled redox conditions, and assayed for biological activity in vitro and in vivo. The renatured rhBMP3-C fusion protein bound tightly to collagen matrices and inhibited DNA synthesis in normal rat calvaria cells and in two out of three human osteosarcoma cell lines tested. Alkaline phosphatase activity was increased in rat calvarial cells and was decreased in osteosarcoma cells in vitro in a dose-dependent manner. Collagen sponges impregnated with rhBMP3-C and implanted subcutaneously in Fischer-344 rats induced dose-dependent dystrophic calcification of the collagen matrix, with no evidence of ectopic bone formation. However, local injection of rhBMP3-C infused in a collagen suspension induced new bone formation on the periosteal surface of rat calvaria. Finally, in a rat cranial defect model, surgical implantation of rhBMP3-C arrayed on either collagen sponges or on porous ceramics coated with Type I collagen exhibited marked osteoinductive properties. Taken together, these results demonstrate the feasibility of engineering and manufacturing targeted-BMPs which exhibit an integral gain-of-function that may be exploited to therapeutic advantage in (i) the enhancement of effective local concentrations, (ii) the prevention of systemic biodistribution and side effects, and (iii) the design of improved osteoinductive matrices.