**2. Bovine amniotic membrane role in regenerative medicine and wound healing**

Regenerative medicine is a branch of medicine concerned with developing therapies that regenerate or replace injured, diseased, or defective cells, tissues, or organs to restore or establish function and structure [17]. Regenerative medicine has become an integrated part in medicine and surgery. It has brought potential therapy possibilities with the aim to restore and improve the function of the damaged tissue or organ [18, 19]. There are many approaches to regenerative medicine, including stem cell, tissue engineering, organ transplantation and biomaterials. Biomaterials are either synthetic or natural material that are used for medical purpose or in contact with biological system. Biomaterials in regenerative medicine are intended to facilitate repair mechanism of wound. Usage of biomaterials in wound healing is applied on biological dressing. Biological dressing prevents evaporative water loss, heat loss, protein and electrolyte loss, and contamination and also permit debridement and develop granulation and epithelialization of wound bed [20].

### *Future of Bovine Amniotic Membrane: Bovine Membrane Application on Wound Healing… DOI: http://dx.doi.org/10.5772/intechopen.99313*

Due to its structure as fetal ectoderm, amnion is considered as a physiological biological dressing [2, 3]. Amnion is favored due to optimal barrier against bacterial colonization and prevention of water loss [21]. Several studies on burn patients showed rapid epithelization of wound bed with minimal graft loss [21–23].

Ideally, the graft should be harvested from the same species (allograft). However, human amniotic membrane harvesting has several limitations. Fresh amnion is proposed to carry several infectious diseases such as hepatitis, AIDS, syphilis and tuberculosis [22]. There are also several difference for each amnion harvested from the same donor. The thickness is different at various site of membrane. The thickness could vary from 0.02 to 0.5 mm. This pattern also relates to its transparency and translucency of the membrane. Multiple attempts from the harvest also creates different thickness from donor harvested near the placenta and another distant from it [24, 25]. Differences acquired from different donors are suggested to be contributed from racial variation, duration of gestation, parity, gravidity, labor term and trial of labor before caesarian section. These factors influence prostaglandin and pro-inflammatory cytokines when transplanted to the recipient [24]. Difficulties in finding donor, long time storage issue also limits the supply [26]. Freeze dried human AM is proposed to be the solution for storage issue. Thomson and Parks in between 1979 and 1980 had studied the preparation of human amnion using sodium hypochlorite 0.025% solution and storaged it at −80° Celcius. There was no negative impact on its clinical benefits [23]. However, legal and religious issues still limit the supply of human amniotic membrane. Bovine amniotic membrane was proposed to help these shortcomings, providing stable supply.

Wound healing is characterized by sequential phases of inflammation, proliferation and remodeling [26, 27]. This process is mediated by many cytokines, growth hormones, and other mediators. Several numbers of studies were conducted resulting that bovine amniotic membrane may exert its therapeutic effects from inflammation to scarring process. Amniotic membrane contains proteinase inhibitors which inhibits migration of polymorphonuclear leucocyte cell to wound bed [28]. Polymoprhonuclear cell is one type of neutrophils that is extremely active on wound healing. Activated neutrophils produce several protease which help killing and degrading microbes. Neutropil-activated protease also breaks down extra-cellular matrix that in turn can debride the wound and facilitate cell migration. Nevertheless, excessive amount of neutrophils has negative impact on wound healing, by causing further tissue damage and inflammation [29]. A study by Kim et al. showed less polymoprhonuclear cell infiltration on amnion-membrane covered group thus promoting rapid healing and inhibiting proteolytic damage [30]. Another study by by Shimmura et al. proved that amniotic membrane attracts and traps inflammatory cells such as monocyte/macrophage, CD4(+) T cell and CD8(+) T cell which are responsible for cell apoptosis [31].

Bovine amniotic membrane also contributes on proliferation phase of wound healing. Abundant growth factors found on its membrane are responsible for the stimulation of proliferation [32]. Epidermal growth factor for instance works during proliferation phase (from day 4 postoperative) and promotes wound healing by increasing the rate of epidermal proliferation and also accelerating wound contraction level related to myofibroblast proliferation and collagen deposition [33]. Insulin growth factor, combined with Platelet derived growth factor and fibroblast growth factor increases the proliferation of fibroblast [34]. Fibroblast is crucial on wound proliferation by creating extracellular matrix and collagen structure for wound bed, as well as contracting the wound. Once fibroblast has migrated into the matrix, it changes its morphology and synthesize granulation tissue components [35]. amniotic membrane also has anti-bacterial peptides which makes it ideal for cell proliferation. Expression of inflammatory molecule suggested that amniotic

membrane is one part of the barrier to progression of infection [36, 37]. Bovine amniotic membrane collagen formation is also similar to human amnion according to its viability, diffusion formation and degradation [38].

Proliferation phase is also characterized by angiogenesis, granulation and epithelization. Each of these steps could also be influenced by amnion application. Matrix metalloproteinase (MMP) is one of growth factor expressed on amniotic membrane. It has been implicated in invasive cellular growth [3, 39]. A study by Jeong et al. showed that increased expression of membrane-type matrix metalloproteinase enhanced the activation of MMP-2 and invasion and migration of endhotelial cells which affect the induction of capillary tube formation [40]. Amnion cells secreted substantial amount of angiogenic factors including HGF, IGF-1, VEGF, EGF, HB-EGF and bFGF [41]. However, there are some studies about in vitro anti-angiogenic effects of amniotic membrane. Faraj et al. found that AM conditioned medium reduced proliferation and angiogenesis. This result was proposed to be induced by thrombospondin and tissue inhibitors of metalloproteinase (TIMP 1) and 2 [42–44].

Amniotic membrane also promotes granulation and epithelization. Analysis by Piscatelli et al. from in vitro model showed that wound contraction in fetus was influenced by combination of pro-contraction transforming growth factor-ß1 and anti-contraction epidermal growth factor [45]. Rapid epithelization was histologicallhy confirmed on bovine amniotic membrane-treated wound with thicker collagen bundles. Keratinocyte migration was also observed on wound bed whereas immunohistochemistry staining for angiogenesis and fibroblast were consistent for proliferation phase [26]. The connective tissue of amniotic membrane contains laminin, fibronectin and collagen, which are the main components of basal membrane. A case by Martinez et al. showed that spontaneous epithelization on epidermolysis bullosa was completed in seven days [46].

Other notable clinical effects of bovine amniotic membrane observed are anti scarring, fluid permeability control and tensile strength property. Anti scarring is promoted by anti-inflammation effect of AM and hyaluronic acid found on its membrane [47, 48]. Fluid control on wound healing is essential. Extravasation of fluid in wound contributes in creating wound exudate. The exudate is a marker of the chronic state of injury. Exudate also creates an environment favorable for bacterial proliferation [49]. Amniotic membrane structure remains intact after sterilization. Clinical study by Rejzek et al. has reported heat, fluid and electrolyte loss prevention by amniotic membrane. Oxygen permeability in amniotic membrane was demonstrated by Yoshita et al., all contributing to accelerated healing process [50, 51].

## **3. Application of bovine amniotic membrane in surgery**

Coradetti et al. demonstrated that mesenchymal stem cells could be derive from bovine amnion. Both amnion and amniotic fluid are capable of differentiating into ectodermal and mesodermal lineages. This study further showed the capability of osteogenic, chondrogenic, adipogenic, and neurogenic stem cell usage for bovine amniotic membrane [52]. Thus, bovine amniotic membrane could be used for many surgical applications.

### **4. Ophthalmic surgery**

The first use of amniotic membrane for ophthalmology was documented by de Rotth back in 1940. The conjunctiva defect caused by symblepharon was treated with fetal amniotic membrane. The graft was fixed to the tendon of rectus muscle

### *Future of Bovine Amniotic Membrane: Bovine Membrane Application on Wound Healing… DOI: http://dx.doi.org/10.5772/intechopen.99313*

and was taken in all cases. The study showed that AM has transformation property toward conjunctiva tissue [53]. Since then, there have been many applications for ophthalmic disease ranging from ocular burn, corneal defects, retinal problems, strabismus, and neoplasia. However, most of them used human amnion [54–60].

Proteins expressed on bovine amniotic membrane showed to be abundant in human cornea: including keratocan, decorin, lumican, TGF-β-induced protein ig-h3, and albumin. These proteins are responsible for corneal healing pathways. Numerous signaling pathway responsible for corneal healing are also revealed in the membrane. Selected pathways include integrin signaling pathway, Cytoskeletal regulation by Rho GTPase, Ubiquitin proteasome pathway, WNT signaling pathway, epidermal growth factor (EGF) receptor signaling pathway [11]. Corneal healing was demonstrated on canine corneal erosion with significantly higher proliferation [61].
