**5.4 Tissue engineering and cell-based bone graft**

The interdisciplinary field of tissue engineering seeks to combine the bone graft substitutes to stimulatory effects of growth factors, (bone morphogenetic proteins and osteogenic proteins) to provide structural support and promote more rapid bone growth and healing. The prime aim of the bone tissue engineering approach is to


#### **Table 3.**

*List of some commercially available synthetic materials and their applications.* 

guide and enhance osteogenic differentiation of stem cells into 3D constructs, so that later it can be engineered successfully into applicable bone constructs. The limitation in availability of both allografts and autografts can be addressed to a great extent by tissue engineered bones or healing of fracture critical defects. The tissue engineering approach for bone grafts differ from other approaches, in the fact that in earlier case,


*Application of Bone Substitutes and Its Future Prospective in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.85092* 

#### **Table 4.**

*List of some commercially available polymer-based graft materials and their applications.* 

integration of engineered bone with patients takes place, thus providing specific and more resilient treatment for condition [66]. Further tissue engineering is more convenient in the case of bone regeneration therapies, as osteogenic differentiation can be achieved through multiple stem cell types [67]. The type of scaffolds and cells used for tissue engineering graft is discussed in more detail.

#### *5.4.1 Scaffolds*

One important requirement for bone tissue engineering is the scaffold, which helps in the migration, proliferation, and differentiation of osteogenic cells promoting new bone formation and regeneration [68]. In this regard, it is required that the scaffold must be stable, biocompatible, and biodegradable and should be porous and permeable for cell seeding, nutrient transport, tissue ingrowth, and vascularization. It should also be osteoconductive, osteoinductive, and osseo-integrative in nature. In clinical settings, a wide range of synthetic and natural scaffolds have been explored for bone repair and bone tissue engineering. Broadly, these materials can be categorized as composites, ceramics, and polymers, with each having specific properties and limitations. Type I collagen polymer matrix serves as a good natural material but suffers from low mechanical modulus.


#### **Table 5.**

*Marketed DBM-based bone graft substitutes.* 

One important group of scaffold, which has been extensively used for tissue engineering, is demineralized bone matrix (DBM). DBM is typically produced from allograft bone by the acid extraction method (known as decalcification). Based on manufacturing techniques, DBM may be presented in different forms like sponges, freeze-dried powder, gel, paste, injectable putty, or strips. It is basically a decalcified allograft retaining collagen and noncollagenous proteins [69]. It is osteoconductive as well as osteoinductive but has no osteogenic property due to its processing. Recently, it is being used in conjunction with cancellous/cortical bone chips. The unlimited availability and reduced immune response owing to acidic demineralization give it superiority while it is generally used as a bone graft additive in spine fusions [70]. The types of DBM bone graft substitute which is commercially available are mentioned in **Table 5**.

#### *5.4.2 Cell type involved in bone tissue engineering*

A variety of *in vitro* culture protocols are used in bone tissue engineering, which requires use of growth factors and cytokines along with specialized dynamic bioreactors. It also requires a large scale of bone extracellular matrix-producing cells, primarily the MSCs. In this regard, the adult bone marrow stem cells (BMSCs) are the cells of choice for tissue engineering. The quantity of the isolated stem cells varies between patients and is indirectly proportional to patient's age [71, 72]. Also, the isolated cells should have high biosynthetic activity, expression of osteogenic markers, and right cell phenotype. Another source for cell-based bone tissue engineering is the adipose tissue which consists of adipose stem cells (ASCs). They undergo differentiation into several cell lineages such as osteogenic, chondrogenic, and endothelial. Comparative studies of ASCs and BMSCs have shown that they share their surface antigen expression pattern involving CD44, CD71, CD90, and CD105 [73]. However, there are differences too, such as the BMSCs mark the absence of hematopoietic and endothelial lineage markers [74–76]. Researchers are currently investigating the ability of other multipotent cells such as periosteum, umbilical cord, cord blood, and fetal tissues to be used for bone tissue engineering [77].

#### *5.4.3 In-vitro culturing*

In this approach, bone marrow is harvested from the patient, followed by *in vitro*  culturing and seeding them on scaffolds prior to implantation into the same patient for tissue regeneration. The bone repair process required many cell types. These cell types are involved in many inflammatory and angiogenic responses and play an important role in development of the bone formation mechanisms. They release cytokines and various growth factors like PDGF, BMPs, VEGF, and interleukins

#### *Application of Bone Substitutes and Its Future Prospective in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.85092*

that attract and simulate the mesenchymal stem cells (MSCs), which are directly involved in bone repair. However, cell sourcing for bone regeneration is still a critical issue for cell-based therapies that need to be addressed. Also, for the sake of achieving homogenous growth in 3D environment, a new bioreactor cultivation system with mass transport capabilities has been explored [78].

#### *5.4.4 In-vivo studies on bone graft applications*

 Multiple reports have been published showing not only the application of bone tissue engineering in the case of ectopic bone formation (nonbone environment) [79] but also in orthotropic bone formation (bone environment) [80]. In 1991, Caplan first reported the usage of autologous stem cells for fast and specific skeletal tissue repair. Mesenchymal stem cells (MSCs) or mesenchymal colony-forming cells (CFU-F) were identified as cell type present in the bone marrow, which have the capability to differentiate into different cell types of bone [81]. In 2004, first time successful transplantation in humans was reported, where lamellar bone was regenerated and repaired by infusing periosteum-derived osteoblasts for 90 days [82]. Thus, bone marrow has been recognized as a source of MSCs and other osteogenic cells, with relative ease of cell collection using the aspiration method [83]. Ever since the identification and recognition of MSCs for repair and engineering of mesenchymal tissues like cartilage and bone, new cell types and putative strategies have been explored vastly. Among newly developed cell types for bone tissue engineering, deciduous dental pulp-derived stem cells and adipose derived cells were the prominent one showing osteogenic potential [84]. Successful tissue engineering-based bone regeneration depends on multiple factors like the number of healthy osteogenic cells transplanted, the use of proper scaffold for seeding the cells at the site of regeneration, enough vascularization of repair site, and osteogenic differentiation factors. The report provided by Coventry and Tapper [62] has proved successful regeneration of ectopic bones in rodent models, fulfilling all the above-mentioned requirements using ceramic based scaffolds [85]. Further bone marrow aspirate and plasmid-rich plasma approaches have been explored for facilitating effective tissue engineering-based grafts.

In addition to these materials, research is still continuing to modify the products for the production of graft materials, which possess all the properties of ideal bone graft. A multidisciplinary approach will be required to improve implanted cell survival on biomaterial substrate with addition of cytokines and other growth factors for prompt new bone ingrowths. This will yield a bony union that resembles natural form and structure. However, the unavailability of widely accepted specific guidelines, standardizing minimum standard for bone grafts, is one of the limiting factors. Also, the size of *in vitro* grown tissue grafts is often found to be small, in the case of critical size defects.

### **6. Conclusion and future perspective**

 The challenge of bone loss and other musculoskeletal complications during surgeries is well recognized, and to address the same issue, different grafting substitutes have emerged. These bone regeneration grafts can be broadly categorized as natural and synthetic grafting materials. Autograft and allograft comprise natural grafting substitutes and between these, allografts are gaining ground and more acceptability with time. There have been revolutionary changes during the past decade, which bring allograft as a first alternative after autograft, which were subsequently interchanged and/or replaced by demineralized bone matrix in

 certain circumstances [83]. The selection of ideal bone graft substitute materials is a difficult task and often lacks substantial scientific backing. However, it is a very fast and broad field of investigation. Current trends remarkably increase the use of synthetic bone graft as alternatives. The introduction of cost-effective, biologically improved synthetic materials that owe the property of ideal bone graft addresses the present clinical needs for the optimal treatment. It is not mandatory to use these synthetic materials alone for reconstructive procedures. However, when it used in the right situations and in combination with autologous, allograft, or other synthetics, in combination with growth factors, they give potentially more desirable results.

 With the advancement of biomechanical research, an interdisciplinary field of tissue engineering has emerged. Limitations associated with the autologous bone grafting method can be addressed to a great extent with the help of bone tissue engineering. However, selection of optimized combinations of cells, synthetic materials (scaffolds) and factors will remain a challenging and complex process. One of them is the selection of materials and cells. This may include the materials, which possess appropriate mechanical properties, degradation rates, and chemical functionality; likewise, for stem cells, it should be isolated from patient-specific cells from the appropriate lineage and directed down on a scaffold construct to heal the proper bone tissues. Cheaper and safer alternatives will probably emerge with a better understanding of the inherent ability of material to induce bone formation after bone graft implantation.

Thus, these developments must also be nurtured and monitored by the group of clinicians and researchers with the knowledge of medical necessity, basic biological principles, and commercial practicality. This may be used for the production of versatile, and easy to implement, allowing for bioengineered bone grafts to more quickly make the leap from bench to bedside. This will help to improve the quality of life of pediatric and adult patients suffering from bone disease and/or disorder. This may be helpful for bridging the gap between bone tissue engineering research and its clinical implications.
