Bone, a biomineralized tissue is a fascinating composite from an engineering standpoint, deeming it to be considered nature’s arguably most robust biological structures known for its fracture toughness and strength. [1] The composite is light weight, produced under ambient conditions and composed of organic-inorganic entities (organoapatites). These organoapatites are synthesized inside the body with energy conservation principles which the 21st century manufacturing principles find hard to replicate. This fascination for bone has inspired design ideas on biomimicry, to develop lightweight yet robust structures ranging from cosmopolitan architecture to automobiles. [2] From a biological perspective, bones apart from providing structural support to the human form, is involved in plethora of functions involving mineral homeostasis, blood production, blood pH regulation and houses important progenitor cells of both mesenchymal and hematopoietic origin. In order for the bone to perform its functions, it undergoes continuous destruction termed resorption carried out by osteoclasts and bone formation carried out by osteoblasts. In the adult skeletal tissues, these two important processes are maintained in balance via bone homeostasis, wherein vascularization and innervation play major roles for achieving this orchestrated event. The skeletal tissue responds to dynamic mechanical loading, inflammatory, hormonal and mineral challenges. Yet the skeletal tissue is capable of acting on its own accord by secreting factors that control the function of other tissues such as kidney, pancreas and gonads, [3] behaving like an endocrine organ.
The adult human skeletal system is constituted by 206 bones, each uniquely different from the five classes: namely long bones, short bones, flat bones, irregular bones and sesamoid.
Flat bones in the cranium differ from the long bone in the limbs, while short bones in carpal/
tarsal regions differ from the irregular bones of the pelvis. Regulatory mechanisms during development and evolutionary adaptation to mechanical loading regime governs the formation of these different bones present in the body [4]. This mechanosensitivity can be attributed broadly to two features. Firstly, the complex hierarchical structure of the bone, intricately built through a bottom-up approach is composed of ordered arrays of mineralized collagen fibrils at nanoscale with simplicity in the design language cascading across micro- and macro-scales [5]. This design, observed across these different length scales of bone help in propagation of forces and contribute to its fracture toughness. [1] Secondly, the forces propagated are sensed by osteogenic lineage cells (especially osteocytes) and respond to them through mechanotransductory pathways. [6] Interesting to note is the loading patterns subsequently get
trickled down across the different length scale in the hierarchical bone structure. For instance, at macro scale for a man (weighing 75 kg) the peak tibio-femoral force would be 2.8 times his body weight with an average resultant joint force of ~ 3 N. [7] On the contrary, forces experienced by osteogenic cells (osteocytes) are in the range of 1 to 10 pN, either directly through the immediate extracellular matrices’ (ECM) stiffness or interstitial fluid flow in the canalicular network of the osteon [8]. The latter in turn contributes to the piezoelectric phenomenon noticed in bone due to the convective ion transport occurring as a result of streaming potentials during interstitial fluid flow. [9, 10] Thus, when the bone is dynamically loaded it exhibits distinguished electrical and magnetic fields. The mechanical loading has been found to play a crucial role in maintaining bone homeostasis, wherein if the mechanical stimulation stops, the anabolic function of bone formation ceases and catabolic function of bone resorption is initiated.
Apart from mechanosensitivity, osteogenic cells are also responsive to the physical and chemical nature of the microenvironment. Various cell types in the tissue respond to different physical and chemical cues, triggering signalling cascades within the cells which form the basis for the feedback routine for cell-fate choices. For instance, it has been well characterized that mesenchymal progenitor cells exhibit robust actin stress fibers when they encounter rigid substrates. This cytoskeletal tension is transmitted to the nucleus via linker of nucleoskeleton to cytoskeleton (LINC) complexes to the nuclear lamins and chromatin by mechanical coupling [11]. Focal adhesion (FAs) complexes-controlled ECM adhesion also has been found to evince similar behaviour. Cells encounter different type of ECM in the micro-milieu having adhesion peptides for anchoring different integrin complexes of the FAs. Thus, the spacing and availability of ECM adhesion sites control the cellular spreading, cytoskeletal tension, and ultimately modulating the gene expression profiling. Cells adapting osteogenic fate are influenced by these physical cues from the micro-environment, and as an outcome seen to upregulate RhoA expression to confer lineage commitment. [12] Apart from these physical cues, chemical cues also regulate the osteogenic fate. Cellular signalling is a complex orchestration of events mediated by several biomolecules such as proteins, peptides, lipids, nucleic acids and their complexes. Manipulating these cellular signalling pathways by synthetic small molecules has been a pivotal area of interest for chemical biologists to harness the enormous potential of cells towards therapeutic or disease etiology understanding. For instance, it is a well-established protocol to utilise β-glycerophosphate, ascorbic acid and dexamethasone as supplements in vitro for driving the osteogenic differentiation of mesenchymal stem cells
(MSCs). Here, dexamethasone is a small molecule corticosteroid which targets glucocorticoid receptors thus playing a crucial role in controlling osteogenic fate. [13]
Presently, bone biomaterials used in clinical setting (for bone and joint substitution) have traversed a long way with the fascination of mending bones with different materials. The earliest reported material as early as 1300 BC in Egypt was the use of wooden splinters for mending femur fracture and wooden prosthesis in case of amputation. [14] The orthopaedic field has evolved and so have the biomaterials alongside, and even more so in the last 50 years.
It can be put into perspective and well regarded that biomaterials have evolved through three generations in these last 5 decades chronologically as follows: first generation (bioinert materials); second generation (bioactive and bioresorbable materials); and third generation biomaterials (smart biomaterials which trigger molecular response to harness cell’s therapeutic potential) [15]. The first two generation biomaterials used in orthopaedic settings has been extensively reviewed elsewhere [15-18] and the third-generation biomaterials that have been developed and are in lab setting (yet to be clinically used extensively) have also been reviewed in the recent years [19-21]. A brief summary of the different bone graft substitutes (under broad classification) used thus far and their drawbacks are listed in Table A1.1 (Appendix). The drawbacks in these current generation biomaterials serve as the driving factors to expedite further research in overcoming them. It is imperative to mention here is that there is huge disparity between the volume of scientific papers generated in the domain of bone tissue engineering and the number of new materials that have traversed into the clinical translation domain. For instance, for the search query: (["bone graft" OR "bone scaffold" OR "bone biomaterial"] AND ["bone regeneration" OR "bone tissue engineering" OR "bone defect repair"]), with limits set for year “2015 to 2021” (searched on 16, June 2021), restricting to only “articles”, conference papers in SCOPUS and Web of Science, following the PRISMA’s statement (http://www.prisma-statement.org). In SCOPUS, 1866 documents were listed with 96.3% of documents as research articles and 3.7% of documents as conference papers, while in Web of Science 9183 records were listed with 98.1 % of records as articles and 1.9 % of records as meeting/ conference records. On the contrary for the same search query, only 188 records were listed for clinical trials in Web of Science while only 37 records were listed in MEDLINE (through PubMed). Positive anecdotes from the recent past usher in hope that research in the right direction would eventually yield fruition, which is the hallmark in Larry L. Hench’s Bioglass® episode [22]. The pressing need to aid US military surgeons during 1960s where metal/ plastic orthopaedic prosthesis available at their disposal faced severe
rejection rate. Thus, the first bone bonding biomaterial capable of forming apatite layer between the implant and bone surface was developed and since 1985, Bioglass® has been widely used in clinical setting improving the implant patency drastically. [22]
The focus of this chapter is to revisit different third generation biomaterials on the context of delineating the existing literature into three important aspects: firstly, as briefly introduced earlier osteogenic cells are responsive to their immediate micro-milieu and hence the different physical, chemical cues are integral part for maintaining their lineage commitment and maintenance. Therefore, it becomes pertinent to understand these cellular dynamics to harness them for therapeutic potential. Secondly, using the knowledge gained over the years in modulating cellular response in vitro, insights obtained from developmental, pathological aspects, “cell-instructive” traits can be conferred on to biomaterials to emulate structural, biochemical features inspired by biomimetics of bone. Different stratagems adopted to confer these cell-instructive traits are discussed in detail in forthcoming sections of this chapter.
R e vi ew o f L it er a t ur e
1.2. Understanding the Cellular Perspective - Catering to the Cells