Üç boyutlu yazıcı ile jelatin bazlı yara örtüsü geliştirilmesi
Development of gelatin-based wound dressing with three-dimensional printer
Yazar: ESRA GİRGİN
Danışman: PROF. DR. MAHMUT ÖZACAR ; PROF. DR. OĞUZHAN GÜNDÜZ
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Biyomedikal Mühendisliği Ana Bilim Dalı / Biyomedikal Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Biyoteknoloji = Biotechnology ; Mühendislik Bilimleri = Engineering Sciences ; Polimer Bilim ve Teknolojisi = Polymer Science and Technology

Özet:
Kollajen türevi olan jelatin, yüksek biyouyumluluğa, hemostatik özelliklere, azaltılmış sitotoksisiteye ve antijeniteye sahiptir. Aynı zamanda hücre yapışmasını ve çoğalmasını destekler. Ancak zayıf mekanik özelliklere ve antimikrobiyal aktiviteye sahiptir. Ayrıca jelatin bazlı yara örtülerine antimikrobiyal ajanların eklenmesi, yara örtülerinin in vitro ve in vivo antimikrobiyal aktivitesini arttırır. 
Bu çalışmada ilk olarak jelatin/ ksantan sakızı hidrojeller farklı oranlarda hazırlanarak 3B yazıcıda basılabilirlikleri değerlendirildi. En iyi basılabilir çözelti oranı belirlenerek içerisine 2000 yılı aşkın süredir birçok farklı cilt durumunu tedavi etmek için topikal bir ajan olarak kullanılan salisilik asit (SA) üç farklı miktarda (5, 7, 10 mg) eklenerek AXO A1 (Axolotl Biosystems, Türkiye) 3B yazıcı ile uygun parametrelerde basımı gerçekleştirildi. Basımı gerçekleştirilen ve kurutulan doku iskelelerinin çapraz bağlama işlemi glutaraldehit çözeltisiyle gerçekleştirildi. Çapraz bağlanan iskeleler etiketli petri kaplarına yerleştirilerek daha ileri analizler için saklandı.
Üretilen iskelelerin termal, mekanik, morfolojik, kimyasal özellikleri ve biyouyumluluk özellikleri ayrıntılı olarak değerlendirildi. 
İskelelerin ilaç salım özellikleri, in vitro ortamı taklit eden fosfat tamponunda farklı zaman aralıklarında bir ultraviyole spektrofotometre kullanılarak analiz edildi. Mekanik test sonuçları %4JEL/%1,2KSN/7MG_SA çözeltisinden üretilen yara örtüsünün yaklaşık 2,39 MPa ile en yüksek gerilme mukavemetini gösterdi. Morfolojik analiz sonucu en düşük gözenek yapısının 1014,5 μm ile %4JEL/%1.2KSN/5MG_SA çözeltisinden üretilen iskeleye ait olduğu gözlenmiştir. Antimikrobiyal özellikleri sırasıyla S.aureus ve E. coli kullanılarak karakterize edilmiş ve iskelelerin  antibakteriyel etki göstermediği ve inhibasyon çapı oluşmadığı gözlemlenmiştir. Bu sonuç yara örtülerindeki SA içeriğinin yetersiz kaldığını ve çözeltideki ilaç miktarını arttırarak antibakteriyel aktivitenin ortaya çıkarılabileceğini düşündürmektedir. 
Termal karakterizasyon sonuçlarına göre, tüm yara örtülerinde 50°C civarlarında Tg noktası gözlemlendi. 
Yara örtülerinin biyouyumluluğu ve sitotoksisitesi MTT tahlili ile belirlendi ve in vitro çalışmalar %4JEL/%1.2KSN iskelesinin fibroblast hücre hattında, diğer yapılara göre daha fazla hücre canlılığına neden olduğunu gösterdi.
Bu çalışmanın, jelatin bazlı hidrojelden 3B baskılı yara örtüsü iskelelerinin üretimi için doğru boyut ve parametrelerin bulunmasında faydalı olması beklenmektedir.

Abstract:
The skin is the human body's largest organ. It keeps microbes out of the body and prevents infection while protecting the body from harmful environmental effects. It is also involved in skin homeostasis, body temperature regulation, and immune responses. Skin is an organ that has important functions such as protection, secretion, temperature regulation, and detection of external stimuli. It is made up of the epidermis and dermis. Rapid recovery to restore skin protection remains a major challenge in cases where skin damage is caused by multiple factors such as abrasion, tear, and heat damage, and especially when large areas of skin are damaged. Hemostasis, inflammation, proliferation, and regeneration are the four stages of wound healing. It is also a highly complex and dynamic process that necessitates the collaboration of various cell types, cytokines, and cell signals. 
Various biomaterials, such as hydrogels, films, sponges, and nanofibers, have been developed for use in wound dressings in order to speed up wound healing and prevent serious complications.
Wound dressings are very important in wound care. It is critical to properly evaluate a wound, prevent bacteria, foreign objects, and excessive leakage, and keep the wound moist. Furthermore, an ideal wound dressing should allow the wound area to breathe while also protecting the surrounding healthy tissue. The most appropriate wound dressing should be chosen based on the wound's condition and basic needs. The ideal wound dressing should maintain a moist environment, remove excess exudate, prevent drying, ensure gas exchange, be impermeable to microorganisms, provide thermal insulation, and be particle-free. It should not be toxic to beneficial host cells, provide mechanical protection, be nontoxic, simple to use, and inexpensive.
Recently, three-dimensional printing has become widely used in biomedical research. This rapidly advancing technology provides a programmable and configurable platform for tissue engineering and regenerative medicine. Among wound dressing production methods, 3D printing technology has gained traction in recent years to support personalised drug delivery treatments. This method yields consistent results in terms of providing the pores and shapes required for wound healing. It also entails stacking the required biomaterials and active ingredients on top of one another. Three-dimensional printing technologies have made significant advances in the field of biomaterials, as well as the ability to produce materials that would be difficult or impossible to produce using traditional methods for many applications.
Biomaterials derived from nature, such as gelatin and sodium alginate, are widely used in the creation of 3D-printed scaffolds. Hydrogels with excellent swelling and water retention capacity are used as organic materials in modern three-dimensional printing and scaffold manufacturing. Hydrogels have a wide range of medical applications, including tissue engineering and drug delivery. Nevertheless, hydrogels' low mechanical strength and rapid degradation limit their use as tissue engineering scaffolds.
Because of its gelling ability, gelatin is an intriguing biomaterial for hydrogel production among natural polymers. Gelatin, a collagen derivative, is biocompatible and hemostatic, with low cytotoxicity and antigenicity. Cell adhesion and proliferation are also aided by it. It does not, however, have good mechanical properties or antimicrobial activity. To improve its mechanical properties, it is cross-linked with other polymers. Furthermore, the addition of antimicrobial agents to gelatin-based wound dressings increases their antimicrobial activity in vitro and in vivo. Because of its processability and cost-effectiveness, it is widely used in clinics as wound dressings, medicines, and adhesives, in addition to its hydrogel properties.
Xanthan gum is an exopolysaccharide generated by bacteria of the genus Xanthomonas. It is a microbial high-molecular-weight heteropolysaccharide used in medicine and tissue engineering due to its biocompatibility, non-toxicity, gelling qualities, ease of usage, and low cost. It is also inexpensive and simple to use. They are frequently employed in the pharmaceutical sector as thickeners, suspending agents, and emulsifiers, as well as in the development of biodegradable hydrogels for skin scaffolds, due to their amazing qualities.
Mechanical weakness and dissolution behaviour are the limiting characteristics of gelatin and xanthan gum hydrogels. It may swell greatly when immersed in water due to solvent absorption. When applied as a wound dressing, this trait causes harm. The wound structure cannot be protected due to severe swelling, and it adheres to the wound. To address this issue, hydrogels are cross-linked, which boosts the material's strength, hydrolysis resistance, and dimensional stability while also preventing swelling.
Since ancient times, salicylic acid (SA), the primary metabolite of Aspirin®, has been widely utilised in the treatment of pain, fever, and inflammation. SA, or ohydroxybenzoic acid, is derived from the metabolism of salicin and has been used as a topical treatment to treat a variety of skin diseases for over 2000 years. It is a natural phenolic chemical with anti-inflammatory and antioxidant properties. It is still used to treat hyperkeratotic skin conditions such as warts, calluses, psoriasis, and ichthyosis.
For numerous biological purposes, SA is frequently encapsulated in nanosubstrates or other formulations. Because of its superior biocompatibility and abundance of natural resources, SA wound healing has received a lot of attention in recent years. SA-based polymers show promise in biomedical applications. Because of its high bioavailability and anti-inflammatory properties, it is also utilised in the treatment of burns.
Dry and passive wound dressings have been replaced in recent years by functional wound dressings that prevent the wound area from drying out, encourage healing (interactive), and protect the site from infection. The goal of this study was to develop an enhanced wound dressing for medical applications based on gelatin and xanthan gum, which are natural polymers with high biocompatibility that provide patients with a comfortable and painless therapy. It is hoped that adding salicylic acid to these natural polymers and printing them with a 3D printer may improve the patient's social life. The goal is to create not only a wound dressing but also a design with therapeutic effects. Because of its high biocompatibility and the analgesic, antipyretic, and anti-inflammatory qualities of the salicylic acid it contains, the dressing will cover the skin illness and heal the affected area. As a result, combining salicylic acid, which has been widely utilised from the past to the present, with the 3D printer, which is the future trend, and conducting a study in this manner intends to provide more forward-thinking solutions by integrating all kinds of advantages.
First, gelatin/xanthan gum hydrogels in various ratios were made, and their printability on a 3D printer was assessed in this work. Three different concentrations (5, 7, and 10 mg) of salicylic acid (SA), which has been used as a topical treatment to treat many different skin diseases for over 2000 years, were introduced to the AXO A1 (Axolotl Biosystems, Turkey) 3D printer to determine the best printable solution ratio. The printing was done using the proper specifications. The printed and dried tissue scaffolds were cross-linked using a glutaraldehyde solution. The cross-linked scaffolds were housed in labelled petri dishes for later examination.
The thermal, mechanical, morphological, chemical, and biocompatibility aspects of the manufactured scaffolds were thoroughly investigated. The drug release properties of the scaffolds were examined using an ultraviolet spectrophotometer at various time intervals in phosphate buffer, which mimicked the in vitro environment.
Mechanical, morphological, chemical, and antimicrobial properties were evaluated in detail. When the band gaps of FTIR analyses were examined, the band shifts in the control group (gel-xanthan) confirmed that the carboxylic groups of xanthan gum and the peptide bond parts of gelatin played a role in gelatin-xanthan interactions, and no specific bond was observed for salicylic acid.
 Mechanical test results showed that the 4%JEL/1.2% KSN/7MG_SA dressing had the highest tensile strength of approximately 2.39 MPa. As a result of morphological analysis, it was observed that the lowest pore structure, with 1014.5 μm, belonged to the scaffold produced from the 4% JEL/1.2% KSN/5MG_SA solution.
Their antimicrobial properties were characterised using S. aureus Atcc and E. coli Atcc, respectively, and it was observed that the scaffolds did not show an antibacterial effect and no inhibition diameter was formed. This result suggests that the SA content in the tissue scaffolds is insufficient and that antibacterial activity can be revealed by increasing the amount of drug in the solution. 
According to the thermal characterization results, a Tg point around 50°C was observed in all wound dressings. 
The dressings' biocompatibility and cytotoxicity were determined via the MTT assay, and in vitro studies revealed that the 4%JEL/1.2%KSN scaffold increased cell viability in the fibroblast cell line compared to other structures.
This study is expected to be useful in finding the correct dimensions and parameters for the production of 3D printed wound dressing scaffolds from gelatin based hydrogel.