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Studies in fetal wound healing. V. A prolonged presence of hyaluronic acid characterizes fetal wound fluid.

Midgestation fetal wound healing is characterized by healing without fibrosis or scar formation. The mechanisms that underlie this remarkable process are mediated in part through a fetal wound extracellular matrix rich in hyaluronic acid. In this study a newly developed assay was used to determine the hyaluronic acid levels in fetal and adult wound fluid. Adult wound fluid had a rapid increase in hyaluronic acid, which peaked at 3 days and decreased to 0 by 7 days. In contrast levels of hyaluronic acid in fetal wound fluid increased rapidly and remained significantly elevated for 3 weeks. This prolonged presence of hyaluronic acid in the matrix of fetal wounds creates a 'permissive' wound environment that promotes fetal fibroblast movement and proliferation and inhibits cytodifferentiation. Such a matrix environment promotes healing by regeneration rather than by scarring. This observation has therapeutic implications. The prolonged application of hyaluronic acid or hyaluronate protein complexes to wounds in children or adults may modulate healing in a manner that makes the wounds more fetal-like.

Animals↗

Therapeutic effect of intra-articular injection of high molecular weight hyaluronic acid on osteoarthritis of the knee.

Intra-articular injections of high molecular hyaluronic acid (1% solution) were given at 1- or 2-week intervals to 43 patients with osteoarthritis of the knee. Of the 45 treated knees, 32 (71%) responded favorably. This therapy was effective if the osteoarthritis was of a grade less than moderate; it was not effective in cases with considerable effusion or in those with gross architectural changes. No serious side effects were encountered. We suggest that the beneficial effects of hyaluronic acid may be attributable to improved lubrication of the affected joint, to normalization of synovial fluid production by the action of hyaluronic acid on synovial tissue, and/or to the improvement of the intra-articular environment by the contribution of hyaluronic acid to the rebuilding of the barrier that protects the synovial membrane and articular surface.

Adult↗

Protein adsorption, fibroblast activity and antibacterial properties of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) grafted with chitosan and chitooligosaccharide after immobilized with hyaluronic acid.

Poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) membrane was treated with ozone and grafted with acrylic acid. The resulting membranes were further grafted with chitosan (CS) or chitooligosaccharide (COS) via esterification. Afterward hyaluronic acid (HA) was immobilized onto CS- or COS-grafting membranes. The antibacterial activity of CS and COS against Staphylococus aureus, Escherichia coli, and Pseudomonas aeruginosa was preserved after HA immobilization. Among them, CS-grafted PHBV membrane showed higher antibacterial activity than COS-grafted PHBV membrane. In addition, after CS- or COS-grafting, the L929 fibroblasts attachment and protein adsorption were improved, while the cell number was decrease. After immobilizing HA, the cell proliferation was promoted, the protein adsorption was decreased, and the cell attachment was slightly lower than CS- or COS-grafting PHBV.

Absorbable Implants↗

Hyaluronic acid as a molecular filter and friction-reducing lubricant in the human inner ear.

Immunofluorescence for hyaluronic acid occurred intracellularly in morphologically highly specialized areas in the adult human inner ear, for instance in the cuticular plates of all types of hair cells, at the apposition between outer hair cells and Deiter's cell bodies and in the near-surface area of Hensen's cells. The cytoskeletal organization in these regions is characterized by tightly packed filamentous proteins. Under physiological stimulus these regions undergo micromechanical change, either actively moving (force generation) or passively vibrating with changes in elasticity. Hyaluronic acid might therefore act as a friction-reducing molecular lubricant. In the lateral wall of the cochlea an accumulation of hyaluronic acid occurred in the loose connective tissue of the spiral ligament, in particular close to the stria vascularis. Due to its complex molecular network, hyaluronic acid offers considerable resistance to bulk flow of water and may exclude molecules. The basal cell region of the stria vascularis is thus given additional support to minimize (seal?) the stria vascularis towards all other areas except the endolymphatic space. Here, hyaluronic acid could act as a molecular filter.

Adolescent↗

[Determination of hyaluronic acid in the nucleus pulposis in acute and chronic degenerative intervertebral disk changes].

AIM OF THE STUDY: The hyaluronic acid concentration of the nucleus pulposus is in direct connection with osmotic printing. This examination shoes the free and bound hyaluronic acid content of the nucleus pulposus during acut and chronically degenerative disc modifications. METHODS: 20 humane disc parts of the rungs L4/5 and L5/S1 were examined. The intervertebral disc material were deep-frozen to -80 degrees C directly after discectomy. The determination of the hyaluronic acid was carried out with the HA-test of the company Pharmacia. The examined disc material was treated with isotonic common salt solution (0.9%) and triton X-100 (2%). The investigation material was examined with the RIA-test. The patients were subdivided in 3 groups reference to there different anamnese. The first group included 5 patients with an acut disc herniation and an anamnese time under 1 week. The second group consisted of 8 patients with an disc herniation and an anamnese time of more than 4 weeks with extensive conservative therapy. In the third group, the control group including 7 patients, a spondylodese was performed due to other degenerative disc modifications. RESULTS: The results of the investigation showed significantly more (p < 0.05) free and bound hyaluronic acid in the group with an acut disc herniation. On the other hand, no significant difference appeared between the control group and the patients with an disc herniation of older anamnese (p > 0.05). CONCLUSION: We could show that the nucleus pulposus contains more hyaluronic acid in the case of an acut disc herniation than in case of older degenerative disc modifications. The results shoes important new views for the discussion of the genesis of acut and chronic nucleus pulposus prolapses.

Acute Disease↗

Effect of hyaluronic acid in naturally occurring and experimentally induced osteoarthritis.

Effect of intraarticular injections of hyaluronic acid was evaluated in two groups of horses, using force plates to assess quantitatively the degree of lameness observed. In six horses, chip fractures were created surgically on the dorsomedial aspects of both radial carpal bones. After the horses were trained for 30 days, 40 mg of hyaluronic acid was injected intraarticularly into the limb showing the greater degree of lameness. Horses were worked for 2 more weeks and then were evaluated. A highly significant (P less than 0.01) increase in weight bearing on the treated limb was observed. Ten horses with clinical unilateral front limb lameness, involving a single joint, also were treated with 40 mg of hyaluronic acid. The day after the injection, the horses resumed training and were examined 2 weeks later. At that time, the horses were free from observable lameness, and this observation was corroborated by force plate data. According to their trainers, the horses' performance improved markedly after the injection and remained improved. Seemingly hyaluronic acid was beneficial when injected into the arthritic joints of these two groups of horses.

Animals↗

Hyaluronic acid and its effect on postoperative adhesions in the rabbit flexor tendon. A preliminary look.

The effect of hyaluronic acid in preventing adhesion formation in a surgically prepared rabbit flexor tendon model has been evaluated. Nineteen rabbits had partial tenotomy of the middle digit flexor profundus tendon, eight being treated with hyaluronic acid and eleven with saline. The quality of tendon repair and the quantity of adhesion tissue were graded. Though both the treated and control groups showed significant adhesions, the hyaluronic acid treated tendons showed less adhesion tissue and better sliding properties as compared to the controls. No significant difference was noted in the quality of tendon repair and no adverse effects of hyaluronic acid were seen. It is proposed that additional investigation into the development of a better model of postoperative tendon adhesions and an improved characterization of this model are needed. This preliminary study suggests that hyaluronic acid is effective in reducing the quantity of adhesions after tendon surgery but warrants further evaluation.

Animals↗

[Rapid assay of hyaluronic acid in serum].

A method for measurement of hyaluronic acid (HA) level in serum was developed based on using "hyaluronic acid binding protein" (HABP)-coated polystyrene beads. After the beads and test serum being mixed, the mixture was incubated together with reaction buffer for 2 hours, and then the beads were washed. Subsequently, biotinylated HABP was added to the washed beads and incubated for 1 hour. Then peroxidase-conjugated avidin was added to the mixture and incubated again for 1 hour. After the beads being washed, a substrate solution was added to the washed beads and left for 1 hour. Then the reaction was stopped by adding 2N-H2SO4. The absorbance at 492 nm was recorded. The analytical range of HA in serum by this method was found to be between 10-800 micrograms/l, and the precision of the HA assay (CV%) was between 3.0-8.4 in the "with-in" assay (n = 10), and 4.8-8.9 in the "between" assay (n = 5). The analytical recovery of HA assay was between 92-115%. In this study, the results in screening of the serum HA level in RA patients (n = 107), OA patients (n = 16) and healthy subjects (n = 30) showed that the HA level of RA patients was demonstrated significantly higher than that of healthy subjects and OA patients.

Adult↗

Efficacy of intraarticular hyaluronic acid in patients with osteoarthritis--a prospective clinical trial.

AIM: The goal of this study was to determine whether or not the intraarticular administration of hyaluronic acid can improve functional parameters, such as isokinetic muscle strength or total work and clinical test results in patients with osteoarthritis (OA) of the knee. METHOD: As part of a prospective, controlled study 43 patients with osteoarthritic changes of both knees (radiographic Kellgren stage II-III) were followed in a right/left comparison. The influence of intraarticularly injected hyaluronic acid (20mg hyaluronic acid/2ml Hyalart) on functional and clinical parameters was analysed. We used the isokinetic system Cybex 600 for measuring maximal isokinetic muscle strength and total work. A total of 20 males and 23 females fulfilled the inclusion criteria with an age between 55-78 years and underwent five injections of hyaluronic acid (one injection per week). The injected knee represented the treatment group, while the contralateral knee served as the control. RESULTS: The maximum peak torque of the knee extensors in the treatment group was measured between 57+/-26.15/32.33+/-19.63Nm prior to the injections and 77.17+/-32.54/47.83+/-21.43Nm following the hyaluronic acid therapy (P< 0.01). The analysis of the knee flexors at angular velocities of 60 degrees /s and 180 degrees /s revealed values of 40.44+/-21.58/22.89+/-16.64Nm and 53.55+/-24.26/34.05+/-17.37Nm (P< 0.01) respectively. The evaluation of the total work of the knee flexors and extensors revealed a significant difference (P< 0.01) between the treatment and control group. The Lequesne score was reduced from 13.57+/-1.88 prior to the injections to 7.94+/-2.53 after the treatment (P< 0.01). The pain score was documented with the help of a visual analog scale. The VAS values were reduced at rest from 3.83+/-1.72cm to 1.36+/-1.42cm and during weight bearing from 7.57+/-1.34cm to 3.75+/-1.32cm in the treatment group (P< 0.01). CONCLUSIONS: This controlled prospective clinical trial confirmed that 5 weekly intraarticular injections of HA (Hyalart) in patients with OA of the knee provide pain relief and functional improvements.

Aged↗

The role of hyaluronic acid capsular material of Streptococcus equi subsp. zooepidemicus in mediating adherence to HeLa cells and in resisting phagocytosis.

Hyaluronic acid is thought to be one of the critical virulence factors of Streptococcus equi subsp. zooepidemicus. The present study was designed to study the role of hyaluronic acid capsular material in mediating adherence and to resist the phagocytosis of the host's immune defence. The studies were performed with two encapsulated S. equi subsp. zooepidemicus and two unencapsulated phase variants. The bacteria had been previously isolated from diseased pigs and monkeys in Indonesia. The presence of capsular material was determined using the hyaluronic acid decapsulation test and by electron microscopic studies. Both encapsulated bacteria showed mucoid colonies after cultivation on blood agar, grew with diffuse colonies in soft agar media and reacted negatively in the salt aggregation test. The unencapsulated bacteria grew with small colonies on blood agar, formed compact colonies in soft agar media and reacted positively in the salt aggregation test. Adherence and phagocytosis studies revealed that the encapsulated bacteria adhered significantly more to HeLa cells and were less phagocytosed by murine macrophages compared to unencapsulated bacteria. Pretreatment of the HeLa cells using hyaluronic acid or pretreatment of the bacteria by hyaluronidase decreased the adherence value of encapsulated bacteria. Pretreatment of bacteria with pronase had no effect. The presented results strongly indicate that the hyaluronic acid capsular material contributes to adherence properties of S. equi subsp. zooepidemicus and might help the bacteria to resist phagocytosis by macrophages.

Animals↗

Providing an environment for reparative dentine induction in amputated rat molar pulp by high molecular-weight hyaluronic acid.

To study provision of this environment, wound healing was examined by light and electron microscopy following pulp amputation and direct capping with hyaluronic acid. Molar pulps of female Sprague-Dawley rats were mechanically exposed and directly capped; the cavities were then restored with glass-ionomer cement. As an experimental control. Calvital (a commercial preparation of calcium hydroxide paste) was used as the direct pulp-capping agent in other rats, and its effect on pulp healing was compared with that of hyaluronic acid. In hyaluronic acid-treated pulps, at 2 days after amputation, wound surfaces were covered with blood and fibrin clots and inflammatory cells such as neutrophils and macrophages. At 1 week, differentiation of fibroblastic and odontoblast-like cells was observed beneath the wound layer; and odontoblast-like cells produced globular calcified nodules along the existing dentine walls. At 2 weeks, a layer of reparative dentine had been formed by odontoblast-like cells over the dentine walls. Between 30 and 60 days, the formation of reparative dentine had extended throughout the pulp chamber. The healing observed after direct capping with hyaluronic acid was, except for the formation of dentine bridges, similar to that seen after Calvital capping. These results suggest that high molecular-weight hyaluronic acid can provide an environment suitable for reparative dentine formation through mesenchymal cell differentiation during healing of the amputated dental pulp.

Animals↗

A comparison of the effect of hyaluronic acid versus gentamicin on corneal epithelial healing.

PURPOSE: An animal model study was conducted to compare the efficacy of recurrent topical applications of hyaluronic acid and gentamicin ointment for the treatment of noninfected, mechanical corneal erosions. METHODS: An artificial, controlled wound of identical size and depth was inflicted to the corneas of three groups of rabbit eyes in order to measure their healing rates. One group was treated with hyaluronic acid (10 eyes) while the second group received gentamicin ointment (10 eyes). The third group remained untreated and served as the control (10 eyes). The rate of re-epithelisation was measured at 8-h intervals until complete re-epithelisation was observed. After complete wound closure, the rabbits were killed, and comparative histological examinations were performed. RESULTS: Rabbit eyes treated with hyaluronic acid showed a significantly enhanced rate of epithelial defect closure compared with untreated eyes and a similar rate to that achieved with gentamicin ointment. In the eyes treated with hyaluronic acid a normal, multilayered epithelium was observed 48 h after complete healing, whereas the gentamicin-treated eyes showed an imperfectly layered epithelium, with irregularity of the cuboidal cells. CONCLUSION: While both hyaluronic acid and gentamicin enhance corneal epithelial healing at comparable rates, our study suggests that hyaluronic acid may have a more favourable effect on the structure of the healing epithelium, and can offer an alternative mode of therapy for non-infectious corneal erosions.

Animals↗

Synthesis of hyaluronic acid by human peritoneal mesothelial cells: effect of cytokines and dialysate.

OBJECTIVE: To assess effects of the inflammatory cytokines (IL-1-beta, TNF-alpha, TGF-beta 1) and dialysate effluent on synthesis of hyaluronic acid by human peritoneal mesothelial cells (HMC) in in vitro culture. METHODS: Dialysate effluent was collected after the overnight dwell of Dianeal 1.5% from patients during CAPD training. HMC were obtained from omentum from nonuremic donors or were harvested from the dialysate effluent from CAPD patients. Synthesis of hyaluronic acid was studied on monolayers of HMC, which were deprived of serum 48 hours prior to experiment. Effects of cytokines were tested in a medium with low serum concentration (0.1%) or in medium mixed (1:1 v/v) with the autologous dialysate. Hyaluronic acid level in medium was measured with radioimmunoassay. RESULTS: Cytokines enhanced synthesis of hyaluronic acid by HMC, and the strongest effect was induced by IL-1. Effluent dialysate stimulates synthesis of hyaluronic acid stronger than 10% FCS. Effluent dialysate and IL-1 synergistically enhance synthesis of hyaluronic acid by HMC. CONCLUSION: Effluent dialysate from CAPD patients stimulates production of hyaluronic acid by HMC and acts synergistically with cytokines.

Cells, Cultured↗

CD44 in human placenta: localization and binding to hyaluronic acid.

CD44, a receptor for hyaluronic acid (HA), has been identified in the stroma of stem and terminal chorionic villi of human term placenta. The CD44 glycoprotein antigen, isolated from placenta by affinity to monoclonal antibody (mAb) 50B4, consisted mainly of species of M(r) 85,000 and 200,000. Radiolabelled CD44 bound specifically to HA attached to plastic, predominantly via the M(r) 85,000 species; this binding was inhibited by soluble HA and hyaluronidase. The binding of CD44 to HA was also inhibited by mAb 50B4 and IM7.8.1, which recognize epitopes of cluster I and II respectively, but was not blocked by a polyclonal antibody to peptide 18-30 of the B loop (residues 12-101). These results suggest that the portion of the B loop of CD44 implicated in the binding to HA is between amino acids 31-101 and that epitopes located outside the B loop, such as that recognized by mAb IM7.8.1 (between residues 132-215), contribute to this interaction. The presence of a functional CD44 molecule in the human term placenta suggest a role for this molecule in situ in the stabilization and orientation of HA network important in the maintenance of the structural integrity of the placenta.

Antibodies↗

Hyaluronic acid salt--a mechanoelectrical transducer.

Hyaluronic acid transduces a very gentle pressure into an electrical potential. Such pressure, depending on its direction, changes the optical rotary dispersion properties of the salt, either increasing the rotation in the direction already shown by the unpressured salt or changing and increasing the rotation in the opposite direction. These finding have implications for understanding the function of the cochlear and vestibular fluids, renal function, and the approximation to frictionless motion of normal joints.

Chlorides↗

Hyaluronic acid production and CD44 expression in cultured dermal fibroblasts of patients with non-insulin-dependent diabetes mellitus with and without chronic ulcers on the lower extremity.

It is well known that hyaluronic acid and its principal receptor, CD44, are implicated in the regulation of the tissue repair process, but their role in the formation of chronic diabetic ulcers has not been studied. Hyaluronic acid metabolism and CD44 expression are regulated by lactate, where their increased production is considered to affect the properties of fibroblasts in non-insulin-dependent diabetes mellitus. The aim of our work was to investigate the possible role of hyaluronic acid and CD44, and their regulation by lactate, in the abnormal wound healing of diabetes. Fibroblasts were derived from uninjured skin from four non-insulin-dependent diabetic patients with ulcers and four without ulcers; and from four healthy age-matched volunteers. We observed that diabetic fibroblasts of both groups produced more L-lactate ( approximately 30%) and incorporated more (3)H-glucosamine into the medium hyaluronic acid ( approximately 28%) than controls. Fibroblasts of the diabetic group with ulcers, unlike those of the group without ulcers, showed significant increases in the high molecular weight hyaluronic acid accumulation in the pericellular matrix (30.5%, p < 0.01) and CD44 expression (27.0%, p < 0.05). Exogenous L-lactate dose-dependently, and equally for all fibroblasts lines, stimulated the accumulation of medium hyaluronic acid (3.7-fold) and CD44 expression (1.5-fold). However, fibroblasts from diabetic patients with ulcers were more (1.4-fold) sensitive to L-lactate in terms of CD44 expression, and responded to L-lactate by the increased accumulation of high molecular weight hyaluronic acid in the pericellular matrix (32.1%, p < 0.01). We propose that specific properties of fibroblasts from diabetic patients with ulcers may be involved in the increased susceptibility of these patients to chronic ulceration.

Aged↗

Synthesis of hyaluronic acid and collagen in skin fibroblasts cultured from patients with osteogenesis imperfecta.

Collagen and hyaluronic acid syntheses were studied in skin fibroblast cultures from patients with osteogenesis imperfecta and age-matched controls by labeling the cultures either with [3H]proline and separating the collagenous proteins with DEAE- and CM-cellulose chromatographies, or double-labeling the cultures with [3H]glucosamine and [14C]glycine and separating radioactive hyaluronic acid from glycoproteins and sulphated proteoglycans by DEAE-cellulose chromatography. The activities of the cell layer hyaluronate synthesizing enzyme complex (hyaluronate synthetase) were also determined. The osteogenesis imperfecta cultures were classified into three variants on the basis of type III collagen synthesis. Type III collagen amounted to approx. 40--50% from total collagen in the first variety and approx. 25--30% in the second variety. No difference was noted in the ratio of type III collagen to total collagen in the third variety in comparison with control cultures. The radioactivities of 3H-labeled hyaluronic acid in DEAE-cellulose chromatograms were compared with those of the 14C-labeled proteins. The ratios ranged 9.2--17.3 in the cultures from the patients and 4.6--8.8 in the control cultures. Hyaluronate synthetase activities were 1.3--2.0-fold higher in the osteogenesis imperfecta cells than in their controls. Increased hyaluronic acid synthesis in skin fibroblasts correlated with the severity of the disease but not with the increase in type III collagen synthesis.

Adolescent↗

Filling the periorbital hollows with hyaluronic acid gel: initial experience with 244 injections.

PURPOSE: To review our initial experience using hyaluronic acid gel (Restylane) as a filler to treat the periorbital hollows. METHODS: This is a retrospective, anecdotal case review of 244 cosmetic hyaluronic acid gel injections in 155 patients. An average volume of 0.9 ml per injection session was used in an individualized pattern that variably included the orbital rim hollow, zygomatic hollow, septal confluence hollow, and eyebrow and cheek fat pad. To achieve smooth contours, a layered, feathered threading technique was used, placing the filler deep to the orbicularis. Hyaluronidase injections were used in 11% of patients at follow-up visits to "dissolve" some of the filler to reduce contour irregularities. RESULTS: One hundred eight of 121 (89%) patients with follow-up visits were satisfied with the cosmetic improvement after hyaluronic acid gel injections. For maintenance, the interval to second injection averaged 6.5 months. Side effects included lumps or contour irregularities (11%), bruising (10%), color change (7%), and fluid (15%). Twelve patients were unsatisfied and were not interested in additional injections: 5 with malar fluid, 3 with lumpy irregularity, and 3 with color change. CONCLUSIONS: Complex 3-dimensional contours and thin skin over bone render periorbital filling difficult. However, with individualized planning and with care taken to create smooth, feathered contours, it is possible to achieve acceptable improvement. We found that most patients considered themselves improved cosmetically, despite occasional side effects including contour irregularity or lumps, bruising, color change, and fluid accumulation. Patients with very thin skin, preexisting color problems, or preexisting eyelid fluid may not be good candidates for periorbital filling with hyaluronic acid gel. The effect of the filler is temporary, of course, and we counsel patients to anticipate maintenance injections at 6- to 12-month intervals.

Adult↗