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Keywords:

  • hyaluronic acid;
  • fibroblast;
  • keratinocyte;
  • proliferation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interests
  9. References

Background

Intradermal application of hyaluronic acid (HA) in varying chain length and cross-linking density is used routinely for hydrodynamic volume replacement of the extracellular matrix to reduce the clinical effects of aging.

Objectives

In vitro data show that via receptors of the hyaladherin group hyaluronic acid has additionally direct or indirect effects on cells. In the case of native noncross-linked HA, it has been proved that the proliferative and metabolic activity of cutaneous fibroblasts can be increased. The aim of this study was to investigate whether these effects can be proved also for cross-linked HA and how these effects can be quantified for different preparations.

Materials and Methods

The effect on proliferative activity in cultures of native cutaneous fibroblasts and keratinocytes was investigated for noncross-linked HA, for noncross-linked HA with added glycerol, for HA that was stabilized in the carboxyl and hydroxyl groups per inner esterification, and for HA that was chemically cross-linked by 1,4-butanediol-diglycidylether, mixed in small particles in a biphasic compound with native HA, each in different concentrations (0.1, 1.0 and 10.0 mg/mL).

Results

HA that was stabilized in the carboxyl and hydroxyl groups per inner esterification induces the strongest proliferative effect on both cell types. Native noncross-linked HA and chemically cross-linked HA show a rather modest proliferative effect and on fibroblasts only, whereas noncross-linked HA with added glycerol in high concentrations provokes a rather antiproliferative effect.

Conclusions

The data show that HA does induce direct effects on cells depending on type and density of the cross-linkage. The practical relevance in terms of a metabolic filler effect needs to be verified in clinical studies.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interests
  9. References

Hyaluronic acid (HA) and its salts or deprotonized forms (hyaluronates) are linear structured and comparatively long biopolymers.[1] Balazs introduced “hyaluronan” as collective term for the acid and its poly-anions that occur within physiological pH conditions.[2]

HA consists of a large number of basic saccharide units linked in a linear chain by glycosidic bonds: C14H21NO11 with a molecular weight of 3 793 208 g/mol, and the disaccharidic head- and endgroup C14H22NO11 and C14H22NO12.[3] The number of the basic units can reach 10.000 and more. The molar mass of the HA of vertebrates would be approximately 4 million Da. The overall structure based on all intra- and intermolecular interactions of the HA molecules is also referred to as supramolecular structure.[4]

Within physiological conditions, HA is detectable both in a free dissolved form as well as chemically bound in almost all organs of the human body. Because of its large hydrodynamic volume, HA is a substantial component of the extracellular matrix (ECM) of the skin, mainly of the corium. It is the basic substance where cell can proliferate, differentiate, and migrate, and has direct and indirect effects on the cell–matrix as well as cell–cell interactions.[5] Depending on its chain length, HA has also direct regulative effects via intra- and extracellular and membrane-embedded receptors (hyaladherins).[6-9] Therefore, HA works as a signaling molecule for the regulation of inflammatory processes,[10, 11] tumorgenesis,[12] metastasis formation,[13] neoangiogenesis[14] and wound healing.[15] Very important here are the products of the biodegradation of HA, resulting from enzymatic hydrolysis (hyaluronidases)[16] or from elimination.[17, 18] Their functional groups are less masked and therefore show an increased physiological activity.

In the course of extrinsic aging, especially due to UVB radiation, defects within the matrix metalloproteinase 1 cause the proteolytic cleavage of dermal collagen.[19] The reduced synthesis of HA causes alterations that result in a modified dermal morphology[15, 20, 21] with a reduced turgor pressure within the tissue and the formation of wrinkles. In response to this, the intradermal application of nonanimal (produced by fermentation) stabilized HA (NASHA) in varying chain length and cross-linking density is a common procedure to replace the hydrodynamic volume of the extracellular matrix and thus to reduce the clinical effects of aging.[22] In addition, it has been proved that native HA has pro-proliferating effects on cutaneous fibroblasts.[23] The expression of CD44 seems to have a great impact here,[24] although it is not clear whether the effects are caused by HA or rather by its products of biodegradation.[25] There is also a debate on whether HA has an indirect influence on the metabolism of the fibroblasts and their collagen production via biomechanical stretching[26] or cytokine cascades.[27] The pro-proliferative effects of HA have been proved for noncross-linked HA only.[28] It is still unclear whether these effects can be applied also for cross-linked HA and whether these effects are dependent on the type of cross-linkage resp. stabilization. Therefore, various HA produced by fermentation from established cosmetic preparations for intradermal application have been comparatively tested in vitro in this study regarding their impact on the proliferative activity of fibroblasts and keratinocytes.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interests
  9. References

Materials

For the tests, sterile HA produced by fermentation from commercially available derma filler preparations were used: native noncross-linked HA (Hyal® System, Merz Pharmaceutical GmbH, Frankfurt, Germany), native noncross-linked HA with added glycerol (Mesolis® Plus, Anteis S.A., Genf, Switzerland), HA that was stabilized in the carboxyl and hydroxyl groups per inner esterification (Hyal® ACP, Merz Pharmaceutical GmbH, Frankfurt, Germany), and HA that was chemically cross-linked by 1,4-butanediol-diglycidylether, mixed in small particles in a biphasic compound with native HA (Restylane® Vital, Q-Med AB, Uppsala, Sweden). In addition, glycerol (Sigma-Aldrich, purity ≥99%, St. Louis, USA) was tested for comparison. The different concentrations to be tested in vitro were prepared under sterile conditions by dilution of the original preparations with cell medium with a ratio of 0.1, 1.0, and 10.0 mg/mL, respectively. Every individual HA test preparation as well as glycerol in different concentrations was tested against a nontreatment control. Supplementary, the concentration with the least effect of both the native noncross-linked HA as well as of the HA that was stabilized in the carboxyl and hydroxyl groups per inner esterification were tested in combination.

Methods

The incorporation of [3H]-thymidin in the DNA was used as a marker for the proliferative activity of the tested cellular systems. The cellular systems were native cutaneous fibroblasts and native keratinocytes from surgical specimens of juvenile human foreskin. The cells were isolated according to current standard. The cells were cultivated in 24er microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) at 37 °C, 20% CO2 and 80% humidity until confluency of 85% was reached and then exposed to the test preparations for 24 h. Subsequently, 0,5 μCi [3H]-thymidin (Hartmann Analytic GmbH, Braunschweig, Germany) per sample was added. After 1 h the cells were harvested using a cell harvester (Inotech, Wohlen, Switzerland), transferred into scintillator tubes (Ultima Gold, PerkinElmer, Rodgan, Germany) and after further 24 h measured in a liquid scintillation counter (Wallac-ADL GmbH, Freiburg, Germany). For each test setting six cultures were tested independently (n = 6).

Biometric evaluation

The statistical software SigmaStat V 1.0 for Windows was used for biometric evaluation. The statistics were descriptive and were calculated from the results of all independent tests. The box-plot charts show the 5th, 25th, 50th (median), 75th, and the 95th percentile, respectively. After checking the normal distribution and the variance homogeneity, the independent samples were compared using one-way analysis of variance, the Student–Newman–Keuls test or Bonferroni test, and the Kruskal–Wallis test. At a significance level of < 0,05 differences were assessed as statistically significant. Significant differences compared with the control were indicated with “*”.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interests
  9. References

The results for fibroblasts and keratinocytes for each preparation, dependent on the respective concentration as well as including the combination of native noncross-linked HA and stabilized HA, each in concentration with the least effect, are shown in Figures 1 and 2.

image

Figure 1. Box-plot chart of [3H]-thymidin incorporation used a marker for the proliferative activity of native human cutaneous fibroblasts (NHDF) as n-fold of control after 24 h incubation time (= 6). A = 0.1 mg/mL; B = 1.0 mg/mL; C = 10.0 mg/mL; D = 0.1 mg/mL HSYS and 0.1 mg/mL HACP; CONT = control; HSYS = Hyal® System; HACP = Hyal® ACP; COMB = combination of Hyal® System and Hyal® ACP; RESV = Restylane® Vital; MESP = Mesolis® Plus; GLYC = Glycerol; * = statistical significant difference to control (≤ 0.05).

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image

Figure 2. Box-plot chart of [3H]-thymidin incorporation used a marker for the proliferative activity of native human keratinocytes (NHKC) as n-fold of control after 24-h incubation time (= 6). A = 0.1 mg/mL; B = 1.0 mg/mL; C = 10.0 mg/mL; D = 0.1 mg/mL HSYS and 0.1 mg/mL HACP; CONT = control; HSYS = Hyal® System; HACP = Hyal® ACP; COMB = combination of Hyal® System and Hyal® ACP; RESV = Restylane® Vital; MESP = Mesolis® Plus; GLYC = Glycerol; * = statistical significant difference to control (≤ 0.05).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interests
  9. References

The data show that direct effects of HA on the proliferative activity of fibroblasts and keratinocytes are in vitro principally verifiable. However, these effects seem to be dependent on the bioavailability of certain binding domains of the HA. Multiple influences need to be considered here. Besides the type and density of cross-linkage, products of biodegradation are most likely involved in initiating effect cascades. Also, a different expression of individual hyaladherins that depends on the cell type and possibly also on the level of differentiation of cells needs to be considered. The studies presented here do not allow substantiated statements hereto.

Nevertheless, it is evident that besides the well-known hygroscopic effects of intradermal applied HA, other effects do appear by the induction of the proliferative activity of fibroblasts especially. To what extend these effects will have a practical relevance in terms of increasing the collagen synthesis can only be speculated. Based on data given by Fisher et al. 2008[26] and Rock et al. 2010[27] we can assume that the induction of proliferative activity of cutaneous fibroblasts cause clinical relevant metabolic changes within the dermal connective tissue. Clinical studies are necessary to prove, whether and to what extend a metabolic activation of fibroblasts sets in. Based on the available in vitro data, in which commonly intradermal applied concentrations of HA were used, a clinical relevance of these effects can be regarded in the first days after application as very likely.

Besides the common application of HA as a temporary dermal volume replacement filler, another field of application could open up: a metabolic filler function for the longer term.

Further studies are needed to allow substantiated statements for individual preparations. For the preparations tested in this study, it can be said that HA that was stabilized in the carboxyl and hydroxyl groups per inner esterification may be suited to provide this metabolic filler effect.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interests
  9. References

The authors thank Mrs. Sylke Fasshauer and Mrs. Claudia Bruhne for excellent technical assistance. The study was fully sponsored by Merz Pharma GmbH, Frankfurt, Germany.

Conflict of interests

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interests
  9. References

JW received lecture fees by Merz Pharma GmbH. DW and RH declare no conflict of interest.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interests
  9. References
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