1086 D3000 new imprint Word template Vol 13, No 1 (2025) ISSN 2167-8677 (online) DOI 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu Modification of 3D Printed Denture Base Material by Hyaluronic Acid to Improve Healing Immunohistological Effect on MMP2 and VEGF Mustafa Mahmood Jasim, Amnar K. Al-Noori, Ghada A. Taqa College of Den*stry, University of Mosul, Mosul, Iraq Abstract Objec?ves: To evaluate the effect of topical release of different concentra?ons of hyalu- ronic acid from 3D printed denture bases on the healing of skin wounds. Materials and Methods: FiKeen healthy adult male rabbits from New Zealand were randomly split into three groups based on how long they would be kept: 3 days, 7 days, and 14 days. We shaved the skin on the back of each rabbit and then made five cuts in it. The wounds were sutured to allow for healing. The animals were split into three groups based on their treatment type. The first group had their wounds treated with just an incision (control nega?ve), the second group had their incisions treated with a 3D printed specimen under the skin incision without hyaluronic acid (control posi?ve). We added 0.25% hyaluronic acid and a 3D printed denture base specimen to the third incision, and we added 0.5% hyaluronic acid and a 3D printed denture base to the fourth incision. The fiKh cut was treated with 1% HA acid and a 3D printed denture base specimen. AKer that, the biopsies were sent for im- munohistochemistry evalua?on. Results: The data from the treated group showed that hy- aluronic acid (HA) speeds up healing. Conclusion: HA-treated groups, par?cularly those ad- ministered 0.5% and 1% concentra?ons, demonstrated markedly accelerated and more comprehensive epithelial regenera?on. Open Access Cita%on: Jasim MM et al. (2025) Modifica%on of 3D Printed Denture Material by Hyaluronic Acid to Improve Healing: Immunohistological Effect on MMP2 and VEGF. Den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1086 Received: October 19, 2025 Accepted: November 9, 2025 Published: December 12, 2025 Copyright: ©2025 Jasim MM et al. This is an open access ar%cle licensed under a Crea%ve Commons AXribu%on Work 4.0 United States License. Email: ghadataqa@uomosul.edu.iq Introduc)on Hyaluronic acid (HA) is a naturally occurring polymer made up of a linear polysaccharide structure that can degrade spontaneously under certain conditions. It plays a crucial role as a primary component of the extracel- lular matrix (ECM) [1]. This biopolymer is both biocompatible and biodegradable, and it can be found in various biological Fluids and tissues [2]. The beneFicial characteristics of HA have made it an important biopolymer in the biomedical Field [3]. HA is an important biopolymer in biomedicine due to its numerous advantages. It maintains well- preserved structural properties and inter- acts with various ECM proteins and collagen Fibers, facilitating cell adhesion, movement, migration, and growth [4]. Numerous biolog- ical roles in wound healing, including angio- genesis and re-epithelialization, have been observed both in vitro and in vivo through the topical application of HA [5]. It has been used in the healing of the socket after tooth extraction [6]. Tissue regeneration and wound healing are complex biological events that rely on the co- ordinated interaction of growth factors, ex- tracellular matrix (ECM) remodeling en- zymes, and cellular responses [7]. Among the most important mediators involved in these processes are vascular endothelial growth factor (VEGF) and matrix metalloproteinase 2 (MMP2), both of which play essential roles in angiogenesis, ECM turnover, and cell mi- gration [8,9]. VEGF is a potent pro-angiogenic cytokine that stimulates endothelial cell proliferation, ModificaAon of 3D Printed Denture Base Material by Hyaluronic Acid to Improve Healing Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 2 migration, and the formation of new blood vessels. Adequate vascularization is funda- mental for oxygen and nutrient supply to the regenerating tissue, thereby supporting suc- cessful repair. Upregulation of VEGF expres- sion has been strongly correlated with accel- erated healing, whereas impaired VEGF sig- naling is often linked to delayed tissue repair and chronic wounds [10]. MMP2, a member of the gelatinase subgroup of the MMP fam- ily, contributes to ECM remodeling by de- grading type IV collagen and other basement membrane components. MMP2 enables cel- lular migration, angiogenesis, and proper tis- sue remodeling through this activity. Never- theless, excessive or dysregulated MMP2 ex- pression can lead to pathological outcomes such as chronic inflammation, impaired heal- ing, or cancer progression [8]. Material and Methods Fifteen Zealand adult male rabbits were used, 5-7 months in age, weighing 1.25-1.5 kg. These rabbits stayed in the Animal House. The house was prepared for the experi- mental studies in the College of Dentistry at standard-setting with temperature main- tained at 25 ± 2 °C. The feeding of these rab- bits were fed three times daily with a normal diet (lettuce and grass). All rabbits had good health throughout the period of the study. All procedures were carried out per the rules es- tablished by the Research Ethics Committee of the College of Dentistry at the University of Mosul in Iraq, using the code (UOM.Dent. 25/1006). We used Fifteen adult male rabbits from New Zealand that were 5 to 7 months old and weighed between 1.25 and 1.5 kg. The Ani- mal House was set up for the experimental studies at the College of Dentistry, where the rabbits stayed at a temperature of 25 ± 2 °C. They were fed three times a day with a nor- mal diet of lettuce and grass. Three groups of three rabbits were ran- domly chosen from a group of animals. There were Five experimental groups of incisions in the dorsal skin of each rabbit, based on the type of dressing material used (Figure 1). The groups were: Group 1: N=5, negative control. Skin inci- sions did not receive any treatment. Group 2: N=5, positive control. Skin incisions treated with a sample of 3D printed denture base material only. Group 3: N=5, treated with hyaluronic acid 1%. Skin incisions treated with the topical part of 3d printed denture base material combined with hyaluronic acid 1% Group 4: N=5, treated with hyaluronic acid 0.25%. Skin incisions treated with the topical part of a 3d printed denture base material combined with hyaluronic acid 0.25% Group 5: N=5, treated with hyaluronic acid 0.5%. Skin incisions treated with the topical part of a 3d printed denture base material combined with hyaluronic acid 0.5%. Biopsies were collected and subjected to im- muohistopathological examination accord- ing to speciFied time intervals. The animals in each group were randomly split into Five smaller groups, each with three rabbits. These groups were: G1: The animals were put to sleep three days after surgery. G2: The animals were put to death seven days after the surgery. G3: The animals were put to death two weeks after their surgery. Overdose anesthesia killed all the animals. The samples from skin and buccal defects were preserved in 10 percent formaldehyde for 48 hours, subsequently treated with eth- anol alcohol and xylene, embedded and la- beled in parafFin wax blocks, frozen for 24 hours, and then sectioned coronally into 4- micron thick slices using a microtome. A strip of cut tissue with wax at the incision level was then put in a 60 °C water bath, where the tissues were put on a glass slide that had been marked. After that, the slides were de-waxed, stained with hematoxylin and eosin, mounted with DPX, and examined with a light microscope. Results Table 1 shows the median scores of granula- tion tissue (GT) formation in Five experi- mental groups across three-time intervals (Day 3, Day 7, and Day 14), with Five rabbits per group (N=5). The Kruskal-Wallis test was used to assess statistically signiFicant Differences among groups and time points. The scores are expressed as median and in- terquartile Range (IQR). On Day 3, there was a statistically signiFicant difference among the groups (p=0.05). The HA 1% group (G5) exhibited the highest granulation tissue formation, while the neg- ative control group (G1) had no granulation tissue. Capital letters (A, B, AB) reFlect these differences: G5 was signiFicantly higher (A), G1 was lower (B), and other groups were in- termediate (AB). On Day 7, intergroup differences remained signiFicant (p = 0.044). G5 (HA 1%) and G3 (HA 0.25%) showed higher granulation tis- sue scores compared to G1 and G2. This sug- gests an early stimulatory effect of HA on granulation tissue formation, especially at higher concentrations. On Day 14, the differences between groups were still statistically signiFicant (p=0.048). G3 and G4 (HA 0.25% and 0.5%) recorded the highest scores (3), indicating enhanced granulation tissue development. G1 and G2 showed lower scores, suggesting that HA ac- celerated the healing process. For G1 (negative control), there was a statis- tically significant increase in granulation tis- sue formation over time (p=0.05), progress- ing from no granulation on Day 3 to moder- ate levels by Day 14. In G2 (positive control), granulation tissue also increased significantly over the three time points (p=0.05), but the levels remained relatively low compared to HA-treated groups. G3 (HA 0.25%) and G4 (HA 0.5%) showed clear time-dependent increases in granula- tion tissue, with statistically significant changes (p=0.042 and p=0.048, respec- tively). By Day 14, both groups reached peak granulation scores (score 3), indicating strong tissue regeneration. Interestingly, G5 (HA 1%) showed high gran- ulation early (Day 3 and 7), but slightly de- creased by Day 14, possibly due to transition from granulation to tissue remodeling (p=0.05). The most prominent effects were observed in groups treated with 0.25% and 0.5% HA, especially by Day 14. The HA 1% group showed a rapid early response, but granula- tion plateaued or declined slightly, likely re- flecting advanced healing. The differences were statistically significant between groups and within each group over time. Data expressed as median and IQR (Inter- Quartile-Range) (N=3 rabbits) (Kruskal- Wallis test). The difference in capital letters means there are significant differences be- tween groups at p≤0.05. The difference in small letters means there are significant dif- ferences between periods at p≤0.05. Table 2 presents the median histopathologi- cal scores of angiogenesis (new blood vessel formation) in five experimental groups across three-time intervals (Day 3, Day 7, and Day 14). Each group included five rab- bits (N=5), and the Kruskal-Wallis test was used to evaluate statistically significant dif- ferences across groups and over time. Data are expressed as medians and interquartile ranges (IQR). On Day 3, no statistically significant differ- ences were observed between the groups (p=0.078). All groups showed low angiogen- esis activity, with medians ranging from 1 to 2. This suggests minimal early vascular re- sponse to treatment at that time point. On Day 7, the Kruskal-Wallis test indicated a significant difference among groups (p=0.05). The HA-treated groups (G3–G5) showed markedly increased angiogenesis (median=4), while the control groups (G1 and G2) had lower scores (medians=2–3). Capital letters show that these differences are statistically significant between groups, ModificaAon of 3D Printed Denture Base Material by Hyaluronic Acid to Improve Healing Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 3 indicating the pro-angiogenic effect of HA at this stage of healing. By Day 14, the differences remained signifi- cant (p=0.048). The HA 0.25% and 0.5% groups (G3 and G4) maintained the highest angiogenesis scores (median=4), while the negative control group (G1) exhibited lower values. G5 (HA 1%) showed a slight reduc- tion compared to Day 7, possibly indicating a transition from active angiogenesis to vascu- lar stabilization or tissue remodeling. In G1 (negative control), angiogenesis grad- ually increased over time, with significant differences observed between days (p=0.048). However, the increase was mod- erate compared to the HA-treated groups. G2 (positive control) also showed a signifi- cant rise in angiogenesis from Day 3 to Day 14 (p=0.034), suggesting a delayed but no- ticeable natural angiogenic response. G3 (HA 0.25%) and G4 (HA 0.5%) both demonstrated a rapid and sustained increase in angiogenesis, with statistically significant differences over time (p=0.028 for both). These groups had consistently high scores from Day 7 onward, highlighting the effec- tiveness of these HA concentrations in pro- moting vascularization. G5 (HA 1%) also showed early enhancement of angiogenesis (Day 3 to Day 7), followed by a slight decrease by Day 14 (p=0.05). This might reflect earlier maturation or stabiliza- tion of newly formed vessels. The findings demonstrate that hyaluronic acid (HA) sig- nificantly enhances angiogenesis in rabbit skin wounds, particularly at 0.25% and 0.5% concentrations. Although all groups exhib- ited a natural increase in angiogenesis over time, HA-treated groups achieved faster and higher vascular growth, especially by Day 7 and 14. The differences were statistically sig- nificant across groups and within each group over time, emphasizing the therapeutic role of HA in promoting wound healing through enhanced vascularization. The histological evaluation of rabbit skin wound sections across all experimental groups at various time intervals (Days 3, 7, and 14) are shown in Figures 1 to 6. Discussion Additive manufacturing, especially three-di- mensional (3D) printing, has become a new and better way to make prosthetics instead of using heat-polymerized polymethyl meth- acrylate (PMMA) [11]. 3D printed denture bases have several beneFits, such as the abil- ity to integrate digital workFlows, shorten lab time, improve reproducibility, and allow for patient-speciFic customization [12]. The layered printing method is important be- cause it lets you control how bioactive agents are added to the polymer matrix. This is not easy to do with traditional processing meth- ods [13]. This creates new chances to turn denture bases from passive prosthetic de- vices into biofunctional systems that can help keep oral tissues healthy [14]. Adding bioactive substances like hyaluronic acid or bioactive glass speeds up the healing of soft tissue and makes it less painful. This helps patients adjust better [15]. ModiFication of denture base materials by different materi- als to improve physical and biological effect [16-18]. The rabbit was chosen for the skin wound model due to its comparatively extensive skin surface area, white skin, and structural resemblance to human skin, which facili- tated the simultaneous creation of multiple wounds [19]. Histologically, rabbits' buccal mucosa is like humans' oral mucosa. In our study, after day three and day seven, the HA 1% group exhibited the highest granulation tissue formation, while the negative control group had no granulation tissue. Also, the most prominent effects were observed in groups treated with 0.25% and 0.5% HA, es- pecially by Day 14. The HA 1% group showed a rapid early response, but granulation plat- eaued or declined slightly, likely reflecting advanced healing [20]. The wound area and contraction ratio of the groups treated with HA revealed a significant wound healing ac- celeration effect [21]. Previous studies have also shown that the topical application of HA can speed up the wound healing process [22]. HA can speed up the wound healing process in oral ulcer [23]. PDGF can promote the expression of CD44 to enhance cell mi- gration. CD44 serves as the primary receptor for HA. The association and interaction be- tween HA and CD44 regulate numerous in- tracellular signaling pathways that govern cellular biological processes. These encom- pass angiogenesis, cellular migration, prolif- eration, adherence to ECM constituents, and the internalization and degradation of HA [24]. In our study, the level of PDGF in the dif- ferent groups reveals intense positive ex- pression to improve the healing process in days 3, 7, and 14 [25]. On the other hand, MMP2 showed intense positive expression in days 3 and 7, and moderate positive ex- pression in day 14, as previous work [26]. Conclusions We concluded that hyaluronic acid release from 3d printed denture base with different concentrations (0.25%, 0.5%, and 1%), espe- cially 1% HA that had a highly positive effect as immunohistochemical changes in wound healing. Acknowledgements We appreciate the College of Dentistry, Uni- versity of Mosul, for providing its support to this work. Conflict of Interest The authors have no con.licts of interest to declare. References 1. Buckley, C., Murphy, E. J., Montgomery, T. R., & Major, I. (2022). Hyaluronic acid: A review of the drug delivery capabilities of this naturally occurring polysaccharide. Polymers, 14(17), 3442. 2. Biswal, T. (2021). 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The in vitro effect of leukocyte-and platelet- rich fibrin (L-PRF) and cross-linked hyaluronic acid on fibroblast viability and proliferation. South African Dental Journal, 73(6), 395-399. 24. Cyphert, J. M., Trempus, C. S., & Garantziotis, S. (2015). Size matters: molecular weight specificity of hyaluronan effects in cell biology. International journal of cell biology, 2015(1), 563818. 25. Knopf-Marques, H., Pravda, M., Wolfova, L., Velebny, V., Schaaf, P., Vrana, N. E., & Lavalle, P. (2016). Hyaluronic acid and its derivatives in coating and delivery systems: applications in tissue engineering, regenerative medicine and immunomodulation. Advanced healthcare materials, 5(22), 2841-2855. 26. Caley, M. P., Martins, V. L., & O'Toole, E. A. (2015). Metalloproteinases and wound healing. Advances in wound care, 4(4), 225-234. A B C Figure 1. (A) Surgical procedure for skin; defect created by scalpel blade no.15 on the dorsal skin of a rabbit. (B) 3D printed denture sample. (C) Sutured skin. ModificaAon of 3D Printed Denture Base Material by Hyaluronic Acid to Improve Healing Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 5 Table 1. Histopathological scores of the granulation tissue GT among study groups in the rabbit skin. Intervals Groups Day 3 (N=5) Median Day 7 (N=5) Median Day 14 (N=5) Median P-value G1 Negative control group (wound only) 0 (0) B b 1 (0) B ab 2 (0) B a 0.05 G2 Positive control group (wound + acrylic) 1 (1) AB b 1 (1) B b 2 (0) B a 0.05 G3 HA 0.25% group (wound + acrylic+HA) 1 (1) AB b 2 (1) B ab 3 (1) A a 0.042 G4 HA 0.5% group (wound + acrylic+HA) 1 (0) AB b 2 (2) AB b 3 (1) A a 0.048 G5 HA 1% group (wound + acrylic+HA) 2 (2) A a 3 (1) A a 2 (1) AB b 0.05 P-value 0.05 0.044 0.048 Table 2. Histopathological scores of angiogenesis (newly formed blood vessels) among study groups in the rabbit skin. Intervals Groups Day 3 (N=5) Median Day 7 (N=5) Median Day 14 (N=5) Median P-value G1 Negative control group (wound only) 1 (0) A b 2 (1) B ab 3 (1) B a 0.048 G2 Positive control group (wound + acrylic) 1 (1) A b 3 (1) B a 4 (2) A a 0.034 G3 HA 0.25% group (wound + acrylic+HA) 1 (1) A b 4 (1) A a 4 (1) A a 0.028 G4 HA 0.5 %group (wound + acrylic+HA) 1 (1) A b 4 (2) A a 4 (0) A a 0.028 G5 HA 1 group (wound + acrylic+HA) 2 (1) A b 4 (1) A a 3 (1) B a 0.05 P-value 0.078 0.05 0.048 ModificaAon of 3D Printed Denture Base Material by Hyaluronic Acid to Improve Healing Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 6 Figure 1. Immunohistochemistry sections of VEGF from rabbit skin at day 3 period. [A]: Negative control (wound only) group reveals slight positive expression. [B]: Positive control (wound +acrylic) group reveals weak positive expression. [C]: hyaluronic acid 0.25% (wound+acrylic+HA 0.25%) group reveals moderate positive expression. [D]: hyaluronic acid 0.5 % (wound+acrylic+HA 0.5%) group re- veals moderate positive expression. [E]: hyaluronic acid 1% (wound+acrylic+HA 1%) group reveals intense positive expression 400X, scale- bar=100µm. Figure 2. Immunohistochemistry sections of VEGF from rabbit skin at day 7 period. [A]: Negative control (wound only) group reveals weak positive expression. [B]: Positive control (wound +acrylic) group reveals moderate positive expression. [C]: hyaluronic acid 0.25% (wound+acrylic+HA 0.25%) group reveals moderate positive expression. [D]: hyaluronic acid 0.5% (wound+acrylic+HA 0.5%) group reveals moderate positive expression. [E]: hyaluronic acid 1% (wound+acrylic+HA 1%) group reveals intense positive expression 400X, scale- bar=100µm. ModificaAon of 3D Printed Denture Base Material by Hyaluronic Acid to Improve Healing Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 7 Figure 3. Immunohistochemistry sections of VEGF from rabbit skin at day 14 period. [A]: Negative control (wound only) group reveals weak positive expression. [B]: Positive control (wound +acrylic) group reveals moderate positive expression. [C]: hyaluronic acid 0.25% (wound+acrylic+HA 0.25%) group reveals intense positive expression. [D]: hyaluronic acid 0.5 (wound+acrylic+HA 0.5%) group reveals intense positive expression. [E]: hyaluronic acid 1% (wound+acrylic+HA 1%) group reveals intense positive expression 400X, scale- bar=100µm. Figure 4. Immunohistochemistry sections of MMP2 from rabbit skin at day 3 period. [A]: Negative control (wound only) group reveals weak positive expression. [B]: Positive control (wound +acrylic) group reveals moderate positive expression. [C]: hyaluronic acid 0.25% (wound+acrylic+HA 0.25%) group reveals moderate positive expression. [D]: hyaluronic acid 0.5% (wound+acrylic+HA 0.5%) group reveals moderate positive expression. [E]: hyaluronic acid 1% (wound+acrylic+HA 1%) group reveals intense positive expression 400X, scale- bar=100µm. ModificaAon of 3D Printed Denture Base Material by Hyaluronic Acid to Improve Healing Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 8 Figure 5. Immunohistochemistry sections of MMP2 from rabbit skin at day 7 period. [A]: Negative control (wound only) group reveals weak positive expression. [B]: Positive control (wound +acrylic) group reveals moderate positive expression. [C]: hyaluronic acid 0.25% (wound+acrylic+HA 0.25%) group reveals intense positive expression. [D]: hyaluronic acid 0.5% (wound+acrylic+HA 0.5%) group reveals intense positive expression. [E]: hyaluronic acid 1% (wound+acrylic+HA 1%) group reveals intense positive expression 400X, scale- bar=100µm. Figure 6. Immunohistochemistry sections of MMP2 from rabbit skin at day 7 period. [A]: Negative control (wound only) group reveals mod- erate positive expression. [B]: Positive control (wound +acrylic) group reveals moderate positive expression. [C]: hyaluronic acid 0.25% (wound+acrylic+HA 0.25%) group reveals intense positive expression. [D]: hyaluronic acid 0.5% (wound+acrylic+HA 0.5%) group reveals intense positive expression. [E]: hyaluronic acid 1% (wound+acrylic+HA 1%) group reveals moderate positive expression 400X, scale- bar=100µm.