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Different Hyaluronic Acid‐Based Formulations Exert Composition‐Dependent Effects on Human Fibroblasts and Osteoblasts

Year: 2026

Type: article

Abstract: Hyaluronic (HA)-based materials show composition- and concentration-dependent responses. Proper formulation and dosage selection are key for clinical application. Hyaluronic acid (HA) is widely used in dentistry for its role in wound healing [1], and hard [2] and soft tissue regeneration [3]. Several HA-based formulations have been introduced aiming to enhance their therapeutic effects by accelerating wound healing [4] or by interfering with biofilm formation [5]. Despite the increasing availability of HA-based products, there is still no conclusive evidence identifying the most effective formulation for specific indications, their interactions with different cell types involved in wound healing, as well as the contribution of the individual raw components. Therefore, this study aimed to provide a head-to-head comparison on the biological effects of two commercially available HA-based gels (one enriched with polynucleotides—PNHA, and one containing octenidine—oHA) on human gingival fibroblasts (HGF-1) and osteoblasts (MG-63). PNHA (Regenfast, Geistlich Pharma AG, Wolhusen, Switzerland) and oHA (Pocket-X, Geistlich Pharma AG) were tested at varying concentrations (0.02%, 0.2%, and 2%) to assess their effects on HGF-1 and MG-63 viability, metabolic activity, and morphology (details in Supporting Information). In addition, pureHA (Merck, Darmstadt, Germany) and octenidine, OCT (Octenisept, Schülke & Mayr GmbH, Norderstedt, Germany), were evaluated independently at concentrations corresponding to those present in the respective gel formulations, including a range of serial dilutions. Details are presented as Supporting Information. MG-63 and HGF-1 showed different viability patterns when cultured with PNHA and oHA (Figure 1a,b). MG-63 viability was comparable among all tested PNHA concentrations, for all experimental time points, while 2% PNHA positively influenced the initial activation of HGF-1, with a higher viability still at T3 and T6 if compared to the 0.02% group. For both cell types, there was a marked difference between the two biomaterials tested at all the selected dilutions at T1, T3, and T6. By contrast, oHA groups showed a constantly low activity, thus suggesting a potential toxic effect of some components. Viability analysis findings are corroborated by proliferation and cell metabolic activity data (Figure 1c,d). The metabolism of both cellular lines maintained the same pattern over time and at the different concentrations of biologic factor. oHA negatively influenced the cells, while PNHA showed positive results, with significant differences between the two biomaterials at all time points and concentrations. The analysis of the individual oHA components (Figure 1f–j), pureHA and OCT, at the same relative concentrations confirmed the initial findings, showing a marked toxic effect of pure OCT at all tested concentrations in both cell lines, with no significant differences. In the presence of pureHA, MG-63 proliferation seems dose-dependent at T1/T3; however, values converged at T6, although remaining lower than the control samples (reference value at 100%). Conversely, pureHA enhanced HGF-1 proliferation during the culturing period, with viable cell numbers comparable to or higher than control. Metabolic activity analysis revealed residual activity in OCT-exposed cells, which progressively declined over time in parallel with increasing cell death, while pureHA treatment was comparable to the control, except for 0.8% pureHA, which was consistently lower (p < 0.0001) for both cell types. Calcein-AM staining (Figure 1e,j) confirmed viability and morphology outcomes. PNHA supported well-adhered, healthy osteoblasts and fibroblasts, whereas oHA impaired cell adhesion and proliferation. Analysis of oHA components showed severe octenidine-induced cytotoxicity, while pure HA preserved normal morphology, confirming its cytocompatibility. This in vitro study is the first direct comparison between two HA-based commercial formulations that act by inducing distinct, concentration- and composition-dependent responses in human fibroblasts and osteoblasts. PNHA supported fibroblast viability and metabolic activity, whereas oHA caused cytotoxicity. Even acknowledging the partial contribution of HA characteristics [6] (i.e., molecular weight, reticulation, additives, etc.), the obtained results highlight that commercially used HA-based products are not biologically equivalent and provide the first dose-dependent comparison in soft- and hard-tissue cells. Consistently with previous reports [7], HA promoted soft tissues cells proliferation and migration. This is particularly evident when HA is combined with polynucleotides, confirming previous data of a synergistic effect of the two components, as PNHA activates pro-survival and migratory signaling pathways in fibroblasts and epithelial cells [4, 7]. In line with this evidence, PNHA exhibited the most pronounced benefits at 2%, while osteoblastic responses remained largely limited over time. This confirms literature data [8] asserting HA supports osteoblastic viability while exerting only limited proliferative stimulation, and suggests that longer exposure or additional cues may be required for differentiation. In contrast, oHA and OCT were highly cytotoxic. Although the antiseptic properties of oHA may be handy for the treatment of a chronic infection like periodontitis and its use has been successfully documented as adjuvant in pocket irrigation or as mouthwash to inhibit bacterial re-colonization and facilitate plaque control [5], our results underscore that antiseptic-containing formulations may hinder tissues healing. Collectively, these results highlight that both formulation type and dosage are critical determinants of HA biocompatibility. Careful selection of HA-containing biomaterials based on the specific clinical indications is therefore essential to maximize benefits and minimize adverse effects. The results confirm that HA-based products are not biologically equivalent. PNHA enhanced HGF-1 and MG-63 viability and proliferation in a concentration-dependent manner, whereas oHA showed octenidine-related cytotoxicity. This highlights the importance of concentration and formulation in biomaterial selection based on the clinical scenario and specific indication. However, potential discrepancies between in vitro cytotoxicity and clinical performance cannot be ruled out when looking at factors such as dilution effects, tissue barriers, exposure time, and antimicrobial benefit/risk balance in vivo. Hence, future in vivo studies are needed to explore the behavior of HA-based products in the wound healing environment. Caroline Viola Busch: conceptualization, methodology, investigation, data curation, writing – original draft, supervision. Giulia Brunello: conceptualization, methodology, investigation, data curation, writing – review and editing, supervision. Kathrin Becker: conceptualization, formal analysis, resources, data curation, writing – review and editing, project administration. Jürgen Becker: resources, data curation, writing – review and editing, project administration. Elena Calciolari: conceptualization, investigation, resources, data curation, writing – review and editing, project administration. Benedetta Ghezzi: methodology, validation, formal analysis, investigation, resources, data curation, writing – original draft, supervision. We would like to thank Geistlich Pharma AG (Wolhusen, Switzerland) for providing one of the products used in this in vitro study (Regenfast, Mastelli, Italy). The company was not involved in the design of the experimental setup or in the analysis of the results. Open Access funding enabled and organized by Projekt DEAL. This manuscript used NLP (ChatGPT) for proofreading an entirely human-generated text, but no other use of AI was made. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors declare no conflicts of interest. All the data related to the present paper are available upon reasonable request from the corresponding author. Data S1: Supplementary Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Source: Journal of Periodontal Research

DOI: 10.1111/jre.70094

Authors: Caroline Viola Busch, Giulia Brunello, Kathrin Becker, Jürgen Becker, Elena Calciolari, Benedetta Ghezzi