**3. Collagen and gelatin**

Collagen is one of the most prevalent proteins in the connective tissue of animals and consti‐ tutes approximately 25% of total body protein in vertebrates. It therefore is an important bi‐ omaterial in medical, dental, and pharmacological fields. After the immunogens in the collagen molecules are dislodged, collagen has excellent biocompatibilities either *in vitro* or *in vivo*. Collagen is capable of being cross-linked into solid or lattice-like gels. Resorbable forms of collagen have been used to dress oral, skin or some of the other soft tissue wounds, for closure of graft and extraction sites, and to promote healing [12]. During *in vivo* implan‐ tation, collagen irritates slight inflammation accompanying with some scar tissues.

cells for enhanced function via pattern recognition receptor (PRR) engagement. (C) The acute inflammatory response is dominated by the action of PMNs. PMNs secrete proteolytic enzymes and reactive oxygen species (ROS), corroding the bio‐ material surface. Interleukin (IL)-8 released from PMNs enhances PMN influx and priming. In the transition from acute to chronic inflammation, PMNs stop secreting IL-8 in favor of cytokines promoting immigration and activation of monocytes and macrophages. (D) Macrophages are the driving force of chronic inflammation. Constant release of inflammatory me‐ diators like tumor-nekrose-faktor alpha (TNFα), IL-6, and monocyte chemotactic protein (MCP)-1 results in permanent acti‐ vation of macrophages. Fusion-inducing stimuli like IL-4 and IL-13 promote the fusion of macrophages to foreign body giant cells (FBGCs,) which form a highly degradative environment on the biomaterial surface. Furthermore, FBGC promote extracellular matrix (ECM) remodeling and fibroblast activation resulting in excessive fibrosis and biomaterial encapsula‐ tion. (E) Macrophage-derived cytokines and pattern recognition receptor engagement activate dendritic cells (DCs) on the biomaterial surface. Depending on the nature of the stimulus, DCs mature to either immunogenic or tolerogenic subtypes,

Compared with autografts which come from the same bodies and are only available in limit‐ ed amounts, allografts are more readily available and accompany with less risk and postop‐ erative morbidity. The healing times is therefore shorter and less painful for a patient with no second surgical site is required (as there is when an autograft is utilised). Currently, the use of allograft tissues is increasingly popular all over the world, with widespread orthopae‐ dic surgeons and debilitating musculoskeletal conditions. Nearly one tissue/organ donor can save or improve the lives of up to 60 people. Especially, Musculoskeletal Transplant Foundation, the world's largest tissue bank, provides allograft tissue and biologic solutions for ligament reconstruction [8]. Meanwhile bone and soft tissue allografts from the Steri‐ Graft™ line has been in existence for over 13 years and has helped doctors and their patients with over one hundred thousand successful transplantations. Before transplantation, a blood sample from the donor is normally tested in case any infected diseases, such as hu‐

Specially, decellularized tissue/organ matrices derived from allografts have been used since the 1940s to support tissue repair and replacement. Their popularity has grown sharply dur‐ ing the last decade with the advent of tissue engineering [10]. At present, decellularized tis‐ sues/organs have been successfully used in a variety of tissue/organ regenerative medicines. The efficiency of cell removal from a tissue/organ is dependent on the origin of the tissue/ organ and the specific physical, chemical, and enzymatic methods that are used. Each of these treatments affects the biochemical composition, tissue ultrastructure, and mechanical behavior of the remaining extracellular matrix (ECM) scaffold, which in turn, affect the host

Collagen is one of the most prevalent proteins in the connective tissue of animals and consti‐ tutes approximately 25% of total body protein in vertebrates. It therefore is an important bi‐ omaterial in medical, dental, and pharmacological fields. After the immunogens in the collagen molecules are dislodged, collagen has excellent biocompatibilities either *in vitro* or *in vivo*. Collagen is capable of being cross-linked into solid or lattice-like gels. Resorbable forms of collagen have been used to dress oral, skin or some of the other soft tissue wounds,

amplifying or suppressing the inflammatory response [5].

114 Advances in Biomaterials Science and Biomedical Applications

response to the material [11].

**3. Collagen and gelatin**

man immunodeficiency virus (HIV), Hepatitis, and Syphilis [9].

A collagen sponge obtained from Beijing Yierkang Biengineering Development Center Chi‐ na was implanted subcutaneously in rats for time periods up to 8 weeks (Figure 2) [13]. One week after implantation, slight inflammation with some lymphocytes, myofibrils and fibro‐ blasts were observed. The appearance of myofibrils and fibroblasts indicated that scar tissue was developed (Figure 2A). Two weeks after implantation, fibrous tissue was formed with scattered macrophage and lymphocyte cells in the fibrous layer. Newly formed blood ves‐ sels appeared in the implant site while the collagen sponges were completely resorbed (Fig‐ ure 2B). Four weeks after implantation, the thin fiber layer had changed into wavelike scar tissue and tightly connected with the surrounding muscles. Capillaries were evident in the new fibrous scars (Figure 2C). Six weeks after implantation, scar tissue in the collagen sam‐ ples was mature (Figure 2D). Eight weeks after implantation, the wave-like scar tissue in the collagen samples became thinner with some lipocytes and vacuoles (Figure 2E) [13].

Collagen compounds, such as collagen/chitosan, collagen/hyaluronan, have been investigat‐ ed extensively during the past several decades. The biocompatibilities of these compounds depend largely on the incorporated constituents. For example, a corneal collagen crosslinked with riboflavin and ultraviolet radiation-A has been used for keratoconus repair of a 29-year-old woman with some good results [14]. In some instances, it is more competing to use a compound to improve the mechanical properties of the collagen based biomaterials. For example, a porous implantable dexamethasone-loaded polylactide-co-glycolide (PLGA) microspheres/collagen glucose sensors [15] and a mitomycin C (MMC) delivery system (MMC-film), incorporating polylactide (PLA)–MMC nanoparticles in a composite film from blends of collagen–chitosan–soybean phosphatidylcholine (SPC) with a mass ratio of 4:1:1 have been explored with no sign of internal infection and fibrous encapsulation in any ani‐ mals after 20 days of implantation [16].

Gelatin is a mixture of peptides/proteins produced by partial hydrolysis of collagen extract‐ ed from the skin, boiled crushed bones, connective tissues, organs and some intestines of an‐ imals such as domesticated cattle, chicken, horses hooves, and pigs [17]. Gelatin possesses a better biocompatibility than its ancestry collagen. Alloimplants of bone matrix gelatin are ef‐ fective in the treatment of bone defects with a low risk of complication such as rejection or infection [18]. Aqueous gelatin solution is an amorphous natural hydrogel in which cells can be encapsulated, extruded and deposited at desired positions. Unlike collagen hydrogel, gel‐ atin hydrogel holds a special gelation property around 20℃. In Tsinghua University the au‐ thor's own group, this property has been explored extensively for rapid prototyping (RP) (or additive manufacturing) of three-dimensional (3D) complex geometrical structures with computer-aided design channel models [19-24]. Until now, a hybrid hierarchical 3D con‐ struct consisting both synthetic polyurethane PU and natural cell/ gelatin-based hydrogel with interconnected macro-channels has been produced via a double nozzle RP technique at a low temperature (-28℃). These constructs have demonstrated excellent in vivo biocompa‐ tibilities [23,25]. This technique holds the potential to be widely used in the future complex tissue/organ manufacturing areas.

**Figure 2.** Light-microscope evaluation of the tissue response to collagen sponges with hamatoxylin-eosin (HE) stain‐ ing: (A) 1 week after implantation; (B) 2 weeks after implantation; (C) 4 weeks after implantation; (D) 6 weeks after implantation; (E) 8 weeks after implantation. The scale bar indicates a distance of 50μm in (A), (C), and (D), and a 25μm in (B) and (E) [13].

Combination of gelatin microspheres/scaffolds with other biomaterials, such as collagen, al‐ ginate, chitosan, hyaluronan, and fibrin has also been explored extensively. For example, a gelatin microsphere containing basic fibroblast growth factor and preadipocytes, is essential to achieve a engineered fat tissue [26]. A PLGA microparticles containing an anticancer agent paclitaxel was formulated for the treatment of lung cancers [27]. Gelatin hydrogel in‐ corporating hepatocyte growth factor induced angiogenic change around the implanted hy‐ drogel [28]. A silk fibroin/gelatin composite scaffold was implanted into subcutaneous pockets on male Sprague-Dawley rats with a slight inflammation reaction. By day 30, the scaffold had been completely infiltrated and organized by fibroblasts and inflamed cells. The greater the gelatin concentration in the scaffold, the faster the degradation rate [29].
