A Seminor On

Biomimetic nanofibrous scaffolds for bone tissue engineering.

Ravi Naraian PandeyM.Tech (polymer Technology)

2 nd –yearDTU,Delhi.

AbstractsBone tissue engineering is a highly interdisciplinary field that seeks to tackle the most challenging bone related clinical issues. The major components of bone tissue engineering are the scaffold, cells, and growth factors. This paper will focus on the scaffold and recent advancements in developing scaffolds that can mimic the natural extracellular matrix of bone. Specifically, these novel scaffolds mirror the nanofibrous collagen network that comprises the majority of the non-mineral portion of bone matrix. Using two main fabrication techniques, electrospinning and thermally-induced phase separation, and incorporating bone-like minerals, such as hydroxyapatite, composite nanofibrous scaffolds can improvecell adhesion, stem cell differentiation, and tissue formation.

IntroductionBone injuries and defects present a significant clinical problem.Most are caused by trauma and are relatively simple to treat;however, complex breaks and pathological fractures arising frommalformation, osteoporosis, and tumors present a difficult challengeto treat effectively. In serious fractures and defects or inelderly patients, complications such as mal-union or non-union aremore common and prevent the bone from healing naturally. Thecurrent gold standard for those cases is an invasive surgery to alignand stabilize the bone, usually with metallic pins, screws, plates, orrods, but these surgical procedures extend the healing time and canrequire multiple surgeries.

Since all the current techniques have significant drawbacks, tissue engineering holds promise in providing an improved clinical therapy. By combining a scaffold, cells, and growth factors, tissue engineering seeks to create an alternative solution to repair bone injuries. There are several advantages that tissue engineering has. The use of an engineered implant would reduce the need for multiple surgeries associated with the removal of metallic stabilizers and graft harvesting. Fewer surgeries results in a quicker recovery time, lower costs, and reduced risks. The tissue engineering implant can also be integrated into the existing tissue, resulting in a seamless transition between the two. This prevents stress shielding and resorption of the healthy surrounding bone. These advantages can be mostly attributed to the development of biomimetic scaffolds, and that is what this review will focus on.

The goal of the scaffold is to provide a 3D environment for cells and tissue to grow on. For a biomimetic scaffold, the model is the natural extracellular matrix (ECM). The major protein of the ECM is collagen, which arranges into nanofibers ranging from 50 to 500 nm in diameter and controls cell behavior with its architecture . In bone, the basic building block of the ECM is the mineralized collagen I fibrils, with collagen comprising about 90% of the protein . At nucleation points on the collagen fibrils there are highly ordered carbonated apatite crystals (Ca5(PO4, CO3)3(OH)) only a few nanometers thick.

Scaffold fabrication Method1.Electrospinning

2. Thermally-induced phase separation

ElectrospinningThe principle of electrospinning is that an electric field is used to overcome the surface

tension of a polymer solution to shoot a jet of liquid out of a needle toward a conducting collector . The volatile solvent evaporates in the air leaving behind, under the right conditions,

a polymer fiber with a diameter that can range from tens of nanometers to microns . Many parameters affect this process including polymer properties, solvent properties solution flow rate, voltage, distance from needle to collector, and polymer concentration, among others.

The wide range of polymers capable of being electrospun is appealing to bone tissue engineering and gives researchers flexibility in designing nanofibrous scaffolds. Generally,

there are two groups of polymers that are used: synthetic and natural. Synthetic polymers, such as poly(L-lactic acid) (PLLA) , poly(glycolic acid) (PGA) and polycaprolactone (PCL) and among others, provide great flexibility in synthesis, processing, and modification. However,

these polymers lack bioactivity and special care needs to be taken to ensure that newly synthesized polymers are biocompatible. Many natural polymers, on the other hand, have

inherent bioactivity with peptide sequences that affect cell adhesion, proliferation, and differentiation. Collagen gelatin silk and chitosan among others, are commonly used natural

polymers for scaffold fabrication, but care must be taken to prevent denaturation when proteins are used .

Thermally-induced phase separationThermally-induced phase separation (TIPS) takes advantage of the thermodynamic

instability of polymer solutions at certain temperatures. It was applied to the fabrication of tissue engineering scaffolds in the 1990s with the work of several labs

which used a novel TIPS technique to produce a 3D nanofibroous.TIPS is usually a five step process involving polymer dissolution, phase separation

and gelation, solvent extraction, freezing, and freeze drying. When a polymer, such as PLLA, is dissolved in a solvent, it becomes thermodynamically unstable at low temperatures and will spontaneously separate into two phases. Under the right

conditions, the polymer-rich phase forms the nanofibrous matrix and the polymer-lean phase is extracted, leaving behind nanofibers ranging from 50 to 500 nm.

CompositesBone is a mineralized structural tissue. Biomimetic composite scaffolds with a mineral component have been widely explored for bone regeneration. The mineral not only adds to the structural integrity of the scaffold, but it can also be actively osteoconductive. Hydroxyapatite (HA) (Ca10(PO4)6(OH)2) is commonly used because it closely resembles the natural minerals found in bone, but other calcium phosphate (CaP)

variants or bioglass are also used for their biocompatibility.

Tissue engineering applications1. Cell source and stem cell

differentiation.2. Bone tissue formation

Cell source and stem cell differentiation. Along with the scaffold, the choice of cells is important for taking the next step to engineer bone. Primary cells are appealing since they are more differentiated and can be harvested from specific tissues. To illustrate their use, Woo et al. first showed that MC3T3-E1 osteoblasts attached better on nanofibers compared to solid walls by a factor of 1.7 . To explain this, they found that the nanofibrous scaffold selectively enhanced the adsorption of proteins such as fibronectin, vitronectin, and laminin. These proteins could allow the cells to anchor more tightly to the matrix, resulting in a higher number of cells attached. They then went a step further to demonstrate the ability of nanofibers to enhance the osteogenic potential of mouse calvarial osteoblasts . Cells were seeded on nanofibrous scaffolds and solid-walled scaffolds and assayed for their mineral deposition and osteogenic gene expression. After 7 days, cells seeded on nanofibers expressed higher levels of osteocalcin and bone sialoprotein compared to those seeded on solid-walledscaffolds.

Bone tissue formation While controlling stem cell differentiation is necessary, it is just one step on the path to bone regeneration with the aim to form new functional tissue.Seyedjafari et al. seeded hydroxyapatite coated and uncoated electrospun PLLA fibers with human cord blood derived stem cells and implanted the scaffolds subcutaneously into mice. After 10 weeks, scaffolds without hydroxyapatite showed no calcium deposition and were surrounded by a granulomatous inflammatory response while scaffolds with hydroxyapatite showed significant mineralization with little inflammatory response. Additionally, higher order bone structures such as trabeculi and bone marrow were found within the newly formed ectopic bone. Cai et al. also used an electrospun PLLA scaffold, but combined it with a collagenous guided bone regeneration membrane

Using a rabbit tibia defect model, implants were composed of a porous collagen membrane, a nanofibrous PLLA membrane, or a bilayer combining the two. After 3 weeks, the defects treated with the bilayer group were 91% filled with new bone tissue compared to 64% for the nanofibrous membrane alone and just 32% for the collagen membrane alone. After 6 weeks, the new bone formed on the nanofibers was found to contain a high percentage of cortical bone, 86% and 77% for the bilayer and nanofibrous membrane alone respectively, compared to just 46% for the collagen membrane

Conclusion

From the inception of tissue engineering as a field, it has garnered interest from biologists, materials scientists, engineers, and physicians. The interdisciplinary nature has led to rapid expansion and advancement as numerous hurdles are overcome. Arguably one of the biggest hurdles is the fabrication of a biomimetic scaffold. Since the ECM of bone is composed of primarily collagen I nanofibers, techniques were developed to fabricate fibers of similar sized. At first, an old principle of electrospinning was applied very successfully to create nanofibrous meshes. Then thermally-induced phase separation was used and combined with porogen leaching and 3D printing to create more 3D nanofibrous scaffolds. Now, even more techniques have arisen, including molecular self-assembly and bacterial cellulose, to fabricate nanofibrous scaffolds.