912 D3000 new imprint Word template Vol 13, No 1 (2025) ISSN 2167-8677 (online) DOI 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu Histopathological Assessment of Wound Healing in Mice Treated with Helianthus Tuberosus Powder Ali Ismiel Yawir, Layla Sabri Yas College of Den*stry, University of Baghdad, Baghdad, Iraq Abstract Objec5ve: Several medicinal plants have been given significant a?en5on in the medical field due to their effect on wound healing. This study aims to iden5fy the effec5veness of tuberosus helianthus powder in skin wound healing in mice. Material and Methods: Tuberosis helianthus powder was purchased from a commercial supplier (medicinal Korean herbs, prince herb). The powder was stored in a dry, air5ght container at room temperature un5l use. It was applied directly to the incisional wounds at a specific concentra5on and frequency (50 mg twice daily). Thirty-six male mice were randomly assigned to three 5me points (3, 7, and 14 days), with 12 mice per 5me point. Each mouse received two incisional wounds: one on the leV side, serving as the control, and another on the right side, designated as the experimental wound. At each 5me point, 12 mice were euthanized, yielding 24 wound samples (12 control and 12 experimental) per 5me point. In total, 36 control wounds and 36 experimental wounds were analyzed. Three physical parameters were taken, including the number of inflammatory cells, blood vessels, and epidermis thickness. All these parameters were on the dorsal aspect of the thoracol- umbar region, into which tuberous helianthus powder was typically done in the treated day dura5on. Meanwhile, the day dura5ons for control mice were leV without any treat- ment. The mice were euthanized on days 3, 7, and 14 aVer wound healing for histopatho- logical study. Results: The histopathological analysis using hematoxylin and eosin (H&E) staining re- vealed significant differences between the control and treatment groups. On day 3, the control group exhibited pronounced inflammatory responses with extensive inflammatory cell infiltra5on, necro5c 5ssue, and fibrin clot forma5on. In contrast, the treatment group showed early signs of wound healing, such as mild inflammatory response and reepitheli- aliza5on with kera5nized stra5fied squamous epithelium. Throughout the study, the treat- ment group demonstrated faster wound contrac5on, enhanced 5ssue remodeling, and re- duced inflammatory cells compared to the control. By day 14, treated mice displayed ma- ture epidermis and dermal granula5on 5ssue with significant angiogenesis, sugges5ng ac- celerated wound healing. These findings support the poten5al of Helianthus tuberosus powder in promo5ng wound healing. Conclusion: Tuberosus helianthus powder plays an important role in the healing pathway in wound healing. Open Access Cita%on: Yawir AI, et al. (2025) Histopathological Assess- ment of Wound Healing in Mice Treated with Helianthus Tuberous Powder. Den%stry 3000. 1:a001 doi:10.5195/d3000.2025.912 Received: April 18, 2025 Accepted: May 14, 2025 Published: June 27, 2025 Copyright: ©2025 Yawir AI, et al. This is an open access ar%cle licensed under a Crea%ve Commons AUribu%on Work 4.0 United States License. Email: Ali.yamir2206@codental.uobaghdad.edu.iq Histopathological Assessment of Wound Healing in Mice Treated with Helianthus Tuberosus Powder Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 2 Introduc)on The healing of skin wounds is a problematic biological procedure by which skin repairs itself after a wound [1,2]. Wound healing is a natural process that returns the skin or mucosa to its normal state [3]. This proce- dure comprises three highly combined stages: InClammation, tissue proliferation, and remodeling [4]. It includes the activation of inClammatory cells, endothelial cells, keratinocytes, and Cibroblasts [5], along with the mixture and liberation of active media- tors (cytokines and development parame- ters) to adjust tissue repair and recreation [2,6]. These stages and their physiological parts might happen in the suitable series and at certain times to realize the ideal function and the Clexible strength of the wounded po- sition of the source skin [6]. Different meth- ods are utilized to support skin wound heal- ing, such as managing local or systemic anti- biotics or using antiseptic results [7]. The in- creasing of opposing pathogenic against presently utilized simulated medications managers like antifungal [8], antiviral prod- ucts and antibiotics has encouraged interest among several investigators in the determi- nation of novel antibacterial combinations created from plants [9], plant depend on phytochemicals as potential alternatives to antibiotics that are still utilized in modern and classical schemes of drug for the disease treatment [10]. The existence of multiple life-sustaining constituents in plants has en- couraged researchers to concentrate on plants for their extensive healing wounds potential [11] . Currently, plants or chemical combinations derived from plants have been utilized as pharmaceutical product manag- ers for treating skin wounds [12]. Medicinal plants show a vital key in wound healing via several different mechanisms like reducing the bacterial amount, improving angiogene- sis, rushing the deposition of collagen, and rising Cibroblast proliferation [13]. The biological features of medicinal plants affect key actions in mammalian wound healing, displaying a vital character in regu- lating the wound-healing pathway [14]. The knowledge of the medicinal value of plants and other substances and their use goes back to the earliest settlers [15]. They are hence regarded as a basis of several macromole- cules like fatty acids, carbohydrates, and pro- teins [16], which can be utilized as bio- materials for emerging new candidate medi- cations to support wound healing [17]. Around 500,000 plant types exist world- wide, and only 1% have been investigated [18]. Tuberosus helianthus is a sunClower that ap- peared in eastern North America and is ex- tensively distributed in the Middle East, particularly Iraq [19]. This plant is (2–3 mm) long with superCicial leaves and plump tu- bers [20]. As the plant was civilized by Native Americans Cirst, they denoted the plant as sun roots. While the plant was presented to other portions of the world [21], varied standard terms were attributed in several languages [22]. It has been recognized that this type shows several medical activities, such as diuretic, purgative, and bowel tonic impacts [23]. It has also been utilized as a folk medicine for treating bone fractures and cutaneous wounds, and even for releasing pain [24]. Studies have also shown that tu- berous helianthus combinations have anti- inClammatory, antimicrobial, antioxidant, antifungal, anticancer, antipyretic, and anal- gesic impacts [25]. Tuberosus helianthus is rich in fatty acids like plasmatic acid that resemble those cur- rent in the sebaceous glands in the skin [26]. These fatty acids have been recorded to keep strong antimicrobial–disinfectant features in the skin [27]. This plant has been utilized generally as a dietary supplement for pain management, to decrease swelling [23], to increase the immune system, and to treat skin wounds in general medicine. It is an abundant source of bioactive compounds like phenolic acids, coumarins [28], and Cla- vonoids that are recognized to use pharma- cological actions, comprising anti-inClamma- tory, antimicrobial, and antioxidant features [29]. This study aims to identify the effectiveness of tuberosus helianthus powder on skin wound healing in mice. Material and Methods This study was cross-sectional and approved by Baghdad University, Baghdad Dental Col- lege, Iraq—thirty-six adult male mice from April 2024 to July 2024. The mice used in the experiment protocol were from Baghdad University Baghdad Dental College. The sam- ples were treated at the Laboratories of the Animal House Center in the College of Bagh- dad Medicine, Baghdad University. Thirty- six adult male mice will be subjected to the induced wound on the right dorsal region of each mouse. A- Incisional wound on the left side of the dorsal region will be treated daily with normal saline for three different dura- tions (3, 7 and 14 days). B-Experimental group: 36 mice will be subjected to the in- duced incisional wound on the right side of the dorsal region of the mice and will be treated daily with tuberosus helianthus powder for three different day durations (3, 7 and 14 days). A full thickness wound1cm×1 cm in size was established on the right side of the dorsal as- pect of the animal, into which tuberosus he- lianthus powder was topically applied in the treated group. In contrast, the control group was left without any treatment. All samples were excised and Cixed in 10% formalin. Af- ter Cixation, they were processed; block and slides were prepared and then stained in He- matoxylin / Eosin for histological studies [30]. Preparation of tuberous helianthus pow- der: Tuberosus helianthus powder was pur- chased from a commercial supplier (medici- nal Korean herbs, prince herb). The powder was stored in a dry, airtight container at room temperature until use [31]. It was ap- plied directly to the incisional wounds at a speciCic concentration and frequency (50 mg twice daily). Using Helianthus tuberosus in powder form instead of liquid or other prep- arations can have several advantages in wound healing, like Stability and shelf Life, Controlled Dosage, Sustained Release, Direct Application and Enhanced Absorption and coverage [32]. 36 adult male mice were clas- siCied into experimental and control groups (one incision on the left side -the control and one on the right side experimental), with three different day durations (3, 7 and 14 days). Three physical parameters were taken, like number of inClammatory cells, blood vessels, and epidermis thickness. All these parameters were on the dorsal aspect of the thoracolumbar region, into which tu- berous helianthus powder was topically done in the treated day duration. The day du- rations for the control incision were left without any treatment. The mice were eu- thanized on days 3, 7, and 14 after wound healing for histopathological study. Animals: Thirty-six male young adults (7-8 weeks old) Kunming mice weighing 20-25 g were purchased from the animal house at the College of Veterinary Medicine, Baghdad University. The environment in the cages was kept at room temperature of 25 ± 2°C [33], humidity 55-60%, with a 12:12 h light/dark cycle. The animals were fed standard pellets and tap water. We used male mice in this study to reduce the contri- bution of hormonal variation associated with the oestrous cycle of female mice, which can affect inClammatory responses and tis- sue remodeling in wound healing [34]. Wound creation: General anaesthesia was performed with a mixture of 90 mg/kg ket- amine (Alfasan, Holland) and 10 mg/kg xylazine (Rugby-Virbac Laboratories, France) intraperitoneally. The dorsal part of the thoracolumbar area on both sides was surgically prepared, and full-thickness 1 cm×1 cm wounds were created with a sur- gical scalpel (blade No.11) on all the ani- mals. Surgery was performed under a ster- ile condition [35]. The lesions on the left side were left undressed overnight to the environment to grow, Microbial Histopathological Assessment of Wound Healing in Mice Treated with Helianthus Tuberosus Powder Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 3 colonization and proliferation. In contrast, the right side was treated with the powder, and 50 mg of H. tuberosus powder was topi- cally applied twice daily for the injury. The incisional wound was made because it ena- bles precise evaluation of inflammation, fi- broblast activity, and collagen remodeling. It is a reliable and reproducible method of measuring wound closure over time. Also, it simulates surgical wounds, which makes it pertinent for possible Helianthus tuberosus clinical applications, such as post-surgical healing [36]. Sample size calculations: The sample size calculation was done utilizing statistical analyses achieved using the statistical pack- age for social sciences (SPSS) ver. 29 with Microsoft Excel 2021. Results of the com- plete number of mice 36 adult male mice from April 2024 to July 2024. The α error was 0.05, and the power was 0.90 for sample size calculation. Frequency distribution for nominated variables describing the detailed mice with samples was done. The entire test was commonly distributed, as variables are presented by different statistical parameters like mean, standard deviation (SD), and standard error mean. A one-way ANOVA test was used to compare the experimental and control groups for three days, shown at a sig- niCicance level of 0.05. Results This study was cross-sectional and approved by Baghdad University, Baghdad Dental Col- lege, Iraq. Thirty-six adult male mice from April 2024 to July 2024. All mice will be sub- jected to a traumatic incision of 1^1cm, with a surgical blade (no 11) on the right side of the dorsum for each mouse with the applica- tion of tuberous helianthus powder on the wound. The mice were euthanized on days 3, 7, and 14 days after the wound, making for a histopathological study. Using ImageJ software, the histopathological analysis will be achieved on hematoxylin and eosin-stained slides, including the inClamma- tory cells, epithelial thickness, and blood ves- sel numbers. Macroscopic Assessment of Wound Heal- ing: Wound contraction over time was mac- roscopically evaluated as the healing pro- cess of the incisional wounds. Digital photo- graphs of these wounds were acquired at multiple time points (D3, D7, and D14) for both groups as visual estimates of wound closure. In addition, , wound contraction was quantiCied by a vernier caliper to evalu- ate the decreasing surface area of wounds with time. Wound healing was noticeably enhanced in Helianthus tuberosus powder-treated ani- mals compared to untreated animals. Dressed wounds showed earlier signs of closure with lower levels of inflammation and higher reductions in wound size over time. In contrast, the control group dis- played impaired healing with an increased wound size and prolonged inflammation. These results concluded that Helianthus tu- berosus powder could improve wounds' healing response, leading to faster contrac- tion and lower inflammatory responses in the early days. This observation is further supported by representative images at vari- ous time points of wound closure in both groups (Figure 1). Microscopic assessment of wound heal- ing-Histopathological result (Hematoxy- lin & eosin) stain: Figures A through N sum- marize microscopic assessements. Effect of the number of inDlammatory cells results on all day durations: A one- way ANOVA test is used to analyze the effect of the number of inClammatory cells on all- day durations. A p-value >0.05 indicates no signiCicant differences between the number of inClammatory cells results and all-day du- rations. At the same time, P-Value < 0.05 rep- resents the signiCicant differences between the number of inClammatory cells and all-day durations. There was a highly signiCicant dif- ference in the number of inClammatory cells results among all day durations according to P-value=0.000. It can be observed that the highest mean value (98.00) of 3 days of con- trol is attributed to the number of inClamma- tory cells at this duration being very high compared with other day durations. The lowest standard deviation value (0.707) of 14 days (control negative) was due to a slight difference in values among all the inClamma- tory cells at this duration, as displayed in Ta- ble 1. Effect of the number of blood vessels re- sults on all day durations: A one-way ANOVA test is used to analyze the effect of the number of blood vessel results according to all-day durations. There was a highly sig- niCicant difference between the numbers of blood vessel results and all-day durations concerning P-value=0.000, as displayed in Table 2. It can be observed that the maxi- mum mean value (8.20) of 7 days post-treat- ment is attributed to the number of blood vessels at this duration, which is very high compared to other day durations. The mini- mum standard deviation value (0.447) of 14- day durations post-treatment is due to a slight difference in values among the number of blood vessels at this duration. Effect of the thickness of epidermis re- sults on all day durations: A one-way ANOVA test was used to analyze the effect of the thickness of epidermis results according to all-day durations. There was a highly sig- niCicant difference between the thickness of epidermis results and all-day durations con- cerning P-value=0.000, as exhibited in Table 3. It can be clearly shown that the maximum mean value (82.1164) of 3-day durations post-treatment is attributed to the thickness of the epidermis at this duration being very high compared with other day durations. The minimum standard deviation value (0.5254) of 14-day durations (control nega- tive) is due to a slight value difference among all the epidermis thicknesses at this dura- tion. Furthermore, there was no formation around the tissue at this duration. Discussion This study evaluated the histopathological aspects of the treatment of wound healing, emphasizing inClammatory cell inCiltration, vascularization, epidermal thickness and maturity of the connective tissue. The results conCirmed signiCicant differences between experimental groups at different time points using one-way ANOVA followed by post hoc tests, emphasizing the efCicacy of the tested treatment in enhancing the wound healing process [37]. However, on day 3, the treated group had signiCicantly fewer inClam- matory cells (mean = 32.40), indicating that the treatment helped reduce the early in- Clammatory response. Controlled inClamma- tion is essential for wound healing; how- ever, it should not be excessive, since it leads to delayed wound healing [38]. Additionally, the reductions in inClamma- tory cell counts observed in the post-treat- ment groups persisted with time. On day 14, the treated group had the least inClammatory cells (mean = 1.80), suggesting a reduced in- Clammatory response. P < 0.05, highlighting the promise of treatment to drive the less in- Clammatory environment required for wound healing [39]. Vascularization: Angiogenesis—forming new blood vessels—is a key component of wound healing. The data were analysed sta- tistically, and a marked increase in the num- ber of blood vessels in the treated groups vs the control groups was seen (O/L). The treated group had a mean of 8.20 blood ves- sels per HPF (p<0.05) compared to the 6.40 in the control on day 7 post-treatment. In- deed, the treatment-generated difference (p < 0.05) suggests that angiogenesis was en- couraged and allows for the delivery of oxy- gen and nutrients to the regenerating tissue, as well as transitioning the healing process from inClammatory to proliferative [38]. These results also validated the favorable ef- fect of the treatment on vascularization, which would be shown by improvements in the vascular content of the treated groups over the other groups at later time points (days 7 and 14). Histopathological Assessment of Wound Healing in Mice Treated with Helianthus Tuberosus Powder Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 4 Epidermal Thickness: Epidermal thick- ness is a critical indicator of epithelialization, and our results show a signiCicant increase in epidermal thickness in the post-treatment groups. On day 3 post-treatment, the treated group had an average epidermal thickness of 82.12 µm, signiCicantly more signiCicant than the control group, which showed a Ci- brin clot and necrotic tissue in the epidermal layer. The above difference (p<0.05) con- Cirms that the treatment resulted in a faster reepithelization and a thicker epidermal sheet with stronger morphology(40). The treated group still had epithelial messaging by day 14, emphasizing how EGF treatment accelerated wound healing and induced epi- dermal integrity. Maturation of Connective Tissue: An- other very important factor of wound heal- ing is the maturation of connective tissue. Connective tissue type analysis showed sig- niCicant differences in inClammatory cell numbers about each type. The control group at day 3 showed necrotic tissue with many inClammatory cells (mean = 98.00), while the treated groups exhibited more mature gran- ulation tissue intestinal types. Statistical analysis suggested that necrotic connective tissue was signiCicantly associated with more signiCicant inClammatory cell counts than other tissue types, including immature gran- ulation tissue and mature granulation tissue. These had the combined effect of enhancing the shift from necrotic tissue to more gran- ulation tissue, which is crucial in the remod- eling phase of wound healing [41]. Conclusion Wound healing is a complex process that in- volves inClammation, granulation and tissue remodeling. Interactions of different cells, extracellular matrix proteins and their re- ceptors are involved in wound healing and are mediated by cytokines and growth fac- tors [42]. The statistical analysis of the histo- pathological data corroborates the fact that the treatment dramatically accelerates the healing process. The decrease in inClamma- tory cells, increased vascularity, thickened epidermal layer, and collagen maturation suggest that treatment acts on multiple parts of the wound healing cascade. These data corroborate the literature on non-inva- sive therapeutic strategies for regenerative enhancement of cutaneous wound healing [38]. Overall, since the treatment reduced inClammation, promoted angiogenesis, and accelerated healing of the epithelial and con- nective tissues, it may be an effective thera- peutic strategy in wound healing. References 1. 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Histopathological Assessment of Wound Healing in Mice Treated with Helianthus Tuberosus Powder Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 5 32. Lee K-D, Sun H-J, Lee M, Chun J, Shin T- S, Choi KS, et al. Ethanolic Extract of Pancake Mixture Powder Supplemented with Helianthus tuberosus Enhances Antidiabetic Effects via Inhibiting Inflammatory Mediator NO Production. 2022. 33. Albir AA, Al-Kaisy Z. Histological Investigation of the Hepatic Tissue in Mice Induced by Soft Laser with Different Doses. Biomedical and Pharmacology Journal. 2019;12(2):989-92. 34. Sheridan JF, Padgett DA, Avitsur R, Marucha PT. Experimental models of stress and wound healing. World journal of surgery. 2004;28:327-30. 35. Dawood AS, AL-Ghaban N. Histological and Immunohistochemical study of the Effect of Local Application of Moringa Oliefera/Marine Collagen on Bone Healing in Rats. Baghdad University. Mcs, thesis; 2021. 36. 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Journal of Baghdad College of Dentistry. 2015;27(3):58-63. Histopathological Assessment of Wound Healing in Mice Treated with Helianthus Tuberosus Powder Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 6 Figure 1. Macroscopic images of the wound contraction in the treated and control groups. (A) Day 3 control. (B) Day 3 treated. (C) Day 7 control. (D) Day 7 treated. (E) Day 14 control. (F) Day 14 treated. Histopathological Assessment of Wound Healing in Mice Treated with Helianthus Tuberosus Powder Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 7 3 days (control negative) The histopathological Cigures of the skin (control negative) showed marked dermatitis that characterized by marked loss of the epidermis that replaced with thick layer of Cibrin clot followed by thick line of necrotic tissue with marked inCiltration of inClammatory cells (MNCs) (A and B), the dermis revealed dermatitis with severe degeneration and necrosis of dermal collagen Cibers, severe inCiltration of leukocytes and edema (C). A B C 3 days (post- treatment) The histopathological Cigures of the skin dermis revealed marked reepithelization (Proliferation of epithelial cells) and formation of keratinized stratiCied squamous epithelium (D). There were thin line of necrotic tissue with mild inCiltration of mono nuclear leukocytes (MNCs) mainly lymphocytes with little hemorrhage between dermis and epidermis (D and E). The dermis comprised of immature Cibrous connective tissue with furthermore Cibroblasts and no hair follicles or sebaceous glands (F). D E F Histopathological Assessment of Wound Healing in Mice Treated with Helianthus Tuberosus Powder Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 8 7 days (control negative) The histopathological Cigures of the skin (control negative) showed proliferating epithelial cells of epidermis (stratiCied squamous epithelium (G). The dermis revealed immature Cibrous con- nective tissue with marked meshwork of angiogenesis (newly formed blood vessel) and moder- ate inCiltration of leukocytes, the Cigures revealed no sebaceous glands or hair follicles (H). G H 7 days post treatment The histopathological Cigures of the skin revealed marked epidermal hyperplasia that characterized by formation of epidermal papilla (I). The dermis comprised of slightly mature granulation tissue with little inCiltration of leukocytes and marked angiogenesis. The Cigures revealed nor hair follicles and neither sebaceous glands (J). I J 14 days (control negative) The histopathological Cigures of the skin (control negative) showed mature epithelial cells of epi- dermis (stratiCied squamous epithelium) (K). The dermis revealed slightly mature granulation tis- sue with many newly formed blood vessels, there was no inCiltration of leukocytes and the Cigures revealed no sebaceous glands or hair follicles (L). K L Histopathological Assessment of Wound Healing in Mice Treated with Helianthus Tuberosus Powder Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 9 Table 1. The relative frequency of the number of inClammatory cells in each duration using the one-way ANOVA test. No. of inDlammatory cells Day durations N Mean Std. Deviation Std. Error P-value 3 days-control 5 98.00 5.385 2.408 0.000 3 days post-treatment 5 32.40 6.229 2.786 7 days-control 5 19.60 3.362 1.503 7 days-post treatment 5 11.60 1.517 .678 14 days-control 5 3.00 .707 .316 14 days-post treatment 5 1.80 .837 .374 Total 30 27.73 33.825 6.176 Table 2. The relative frequency of the number of blood vessels results in each duration using the one-way ANOVA test. No. of blood vessels Day durations N Mean Standard Deviation Standard Error P-value 3 days-control 5 1.00 1.000 .447 0.000 3 days post-treatment 5 4.00 1.225 .548 7 days-control 5 6.40 .894 .400 7 days-post treatment 5 8.20 1.095 .490 14 days-control 5 1.80 .837 .374 14 days-post treatment 5 3.20 .447 .200 Total 30 4.10 2.695 .492 14 days post treatment The histopathological Cigures of the skin revealed mature epidermis epithelial cells (M). The dermis comprised of mature granulation tissue with marked angiogenesis (numerous blood vessels). The Cigures revealed no hair follicles and neither sebaceous glands and there is normal adipose tissue of hypodermis (N). M N Histopathological Assessment of Wound Healing in Mice Treated with Helianthus Tuberosus Powder Vol 13, No 1 (2025) DOI 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 10 Table 3. The relative frequency of the thickness of the epidermis results in each duration using the one-way ANOVA test. Thickness of epidermis Day durations N Mean Standard Deviation Standard Error P-value 3 days-control 5 .00000 .0000 .0000 0.000 3 days post-treatment 5 82.1164 3.1397 1.4041 7 days-control 5 43.8006 2.4992 1.1177 7 days post-treatment 5 34.6008 1.9686 .8803 14 days-control 5 25.8884 .5254 .2350 14 days post-treatment 5 26.5034 1.9070 .8528 Total 30 35.4849 25.2418 4.6085