Future Directions in Regenerative Medicine


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A novel stem cell delivery system composed of collagen and alginate as the core and shell, respectively, has been developed.


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This fibrous carrier has been shown promising to enable the encapsulation of tissue cells and their delivery into damaged target tissues, including bone with defect tunability for bone tissue engineering [ 87 ]. Similar to collagen, elastin is a key structural protein found in the ECM of most tissues; yet, very little is known about the response of bone cells to elastin or its derivatives. Recently, a novel class of ECM-based composite scaffolds with collagen and a genetically engineered polymer, elastin-like polypeptide ELP has been designed and produced.

By embedding the elastin within collagen scaffolds, it is possible to expect superior mechanical properties and drug release characteristics compared to collagen scaffolds alone. Elastin also enhances osteogenic differentiation of stem cells and regulates cells behavior in vitro [ 88 ].

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Cellulose is an organic compound and an important structural component of the primary cell wall of green plants, many forms of algae, and the oomycetes, and is secreted by some bacteria to form biofilms. Cellulose is the most abundant organic polymer on Earth [ 89 ]. Cellulose is used to make hydrophilic and highly absorbent sponges, beneficial in combination with other materials for bone tissue engineering applications [ 90 ].

Several in vitro and in vivo researches tried to optimize synthetic-based, tissue-engineered scaffolds in order to be useful in bone regenerative medicine. After seeding with human MSCs and osteoblasts, the composite imparted beneficial cellular growth capability and gene expression, and mineralization abilities were well established suggesting its potential application in bone regeneration [ 91 ]. As another strategy, a combination of different polymers has been tried to increase the cell cytocompatibility of the synthetic-based scaffolds. Poly l -lactide and poly caprolactone triol are some examples.

Using such combination, new membranes promoted the rat osteoblastic cell behavior in vitro e.


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Surface modification and coating is another strategy to enhance bioactivity of the synthetic scaffolds. Silica nanoparticles have been applied onto the fiber surface of an interbonded three-dimensional polycaprolactone PCL fibrous tissue scaffold. The nanoparticle layer was found to improve the fiber wettability and surface roughness. Thus, it enhanced osteoblast attachment, proliferation, and alkaline phosphatase activities [ 93 ]. Despite many beneficial characteristics of synthetic materials in bone healing and regeneration, their biocompatibility, biodegradability, and regenerative properties are still suboptimal compared to natural-based scaffolds.

Therefore, many attempts have been made to combine synthetic with natural materials. Recently, poly d , l -lactide- co -glycolide has been combined with a naturally bioceramic hybrid material, nanonized pearl powder, as an osteoinductive material: the scaffold was able to influence osteoblast behavior in vitro [ 94 ]. The benefits associated with polyhydroxyalkanoates PHA in tissue engineering include high immunotolerance, low toxicity, and biodegradability.

Bone regenerative medicine: classic options, novel strategies, and future directions

Collagen has been used with PHA to increase the biocompatibility of the scaffold and to support cell proliferation and osteogenic differentiation in vitro [ 95 ]. There is an increasing demand for an injectable cell-coupled three-dimensional 3D scaffold to be used as bone fracture augmentation material. To address this demand, a novel injectable osteogenic scaffold called PN-COL was developed, using cells, a natural polymer collagen type I , and a synthetic polymer PCL.

This simple yet novel and powerful approach provides a great benefit as an injectable bone scaffold over other non-living bone fracture stabilization polymers, such as polymethylmethacrylate and calcium content resin-based materials. The advantages of injectability and the biomimicry of collagen were coupled with the structural support of PCL nanofibers to create cell-encapsulated Injectable 3D bone scaffolds with intricate porous internal architecture and high osteoconductivity.

The effects of PCL nanofiber inclusion within the cell-encapsulated collagen matrix have been evaluated for scaffold size retention and osteocompatibility, as well as for MC3T3-E1 cells osteogenic activity.


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At structural analysis, this novel bioactive material was chemically stable enough in an aqueous solution for extended periods without using crosslinking reagents, but it is also viscous enough to be injected through a syringe needle. Data from long-term in vitro proliferation and differentiation suggests that PN-COL scaffolds promote osteoblast proliferation, phenotype expression, and formation of mineralized matrix [ 96 ]. Addition of poly ethylene glycol , as a copolymer source, produced more stable and efficient electrospun jets and aided in the electrospun ability of the PLA nanofibers incorporating the nanocrystallites [ 97 ].

Different types of mono-, bi-, and tricalcium phosphate bioceramics and molecules have been extensively used in bone tissue engineering researches and developments [ 68 ]. Hydroxyapatite is a naturally occurring mineral form of calcium apatite. Carbonated calcium-deficient hydroxyapatite is the main mineral of which dental enamel and dentin are composed [ 98 ]. Both the calcium phosphate and apatite forms have wide applications in bone tissue engineering [ 99 ].

Several authors have used such materials alone or in combination with other materials such as collagen, alginate, and chitosan in order to develop new scaffolds and tissue engineering strategies [ 98 — ]. The strategy for inserting HA nanocrystals within the hydrogel matrix consists of making the freeze-dried hydrogel to swell in a solution containing HA microcrystals.

When the composite CMC-HA hydrogel was characterized and seeded with osteoblasts MG63 line, the scaffold with HA enhanced cell proliferation and metabolic activity and promoted production of mineralized extracellular matrix more than that observed for the scaffold without HA [ ]. Sagar et al. At histology and fluorescence labeling, the uniformly interconnected porous surface of the scaffold construct enhanced osteoblastic activity and mineralization. The composite controls the cell behavior to accelerate and trigger osteogenic differentiation in vitro [ 99 ].

A collagen-hydroxyapatite Col-HA composite through controlled in situ mineralization on type I collagen fibrils with nanometer-sized apatite crystals was designed and produced. After culturing the scaffolds with MSCs, the porous Col-HA composites had good biocompatibility and biomimetic properties and supported bone regeneration and formation [ ].

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A combination of collagen and HA has been used in vivo. Recently, a biomimetic collagen-apatite scaffold composed of collagen fibers and poorly crystalline bone-like carbonated apatite nanoparticles was developed to improve bone repair and regeneration. In vivo , the scaffold enhanced new bone formation in mice [ ]. In addition, the effect of resorbable collagen membranes on critical size defects in rabbit tibiae filled with biphasic calcium phosphate has been investigated: biphasic calcium phosphate functioned well as a scaffold and allowed mineralized tissue formation.

Furthermore, the addition of absorbable collagen membranes enhanced bone gain compared with non-membrane-treated sites [ ].

The application of porous HA-collagen as a bone scaffold represents a new trend of mimicking the specific bone extracellular matrix. Application of HA in reconstructive surgery has shown that it is slowly invaded by the host cells. Therefore, implant compatibility may be augmented by seeding cells before implantation. Human primary osteoblasts were seeded onto innovative collagen-gelatin-genipin GP -HAp scaffolds.

The composite is highly porous, enabling osteoblast-like cell adhesion and growth [ ]. Jung et al.

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This strategy leads to superior new bone formation and bone quality compared with bone graft alone. Calcium phosphate ceramics are sought because they can be bone bioactive, so that apatite forms on their surface, facilitates bonding to bone tissue, and is osteoconductive [ ]. In a recent study, the bioactivity of electrospun composites containing calcium phosphates and their corresponding osteogenic activity was investigated.

Four 8-mm-diameter defects were produced in ten rabbits. Three of the defects in each rabbit were separately and randomly filled with one of the three experimental Ca-P ceramic particles, and the fourth was filled with blood clots control. Piccinini et al. Farahpour et al. Velasquez et al. A fully mineralized new bone growing in direct contact with the implants was found under the in vivo conditions. When implanted, they bind to collagen, growth factors, and fibrin to form a porous matrix to allow infiltration of osteogenic cells [ ]. Recently, a novel nanocomposite hydrogel made of collagen and mesoporous bioactive glass nanoparticles with surface amination has been developed [ ].

The addition of bioglass into the collagen hydrogel significantly increases the bioactivity of the scaffold and improves its mechanical properties; this novel strategy would therefore be suitable for bone tissue engineering applications [ ]. Moreover, the bioactive glass foam produced by sol—gel is an osteoinductive material with a network of interconnected macropores necessary for cell colonization.

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It has been shown that bioactive glass can differentiate human adipose-derived stem cells into osteoblasts, in vitro [ ]. Moreover, Gu et al. The scaffolds can guide bone regeneration and have a controllable degradation rate. A combination of glass and HA has also been used in bone regeneration. Fredericks et al.

Healing promotive factors such as growth factors have been extensively used to treat bony defects and for osteoinduction. These factors can regulate vascularization and induce proliferation and differentiation of the osteoblasts and their precursors, so they can be useful in improving the healing processes [ 32 ]. Bone morphogenetic protein-2 BMP-2 is a potent osteoinductive cytokine that plays a critical role during bone regeneration and repair.

In the extracellular environment, sulfated polysaccharides anchored covalently to glycoproteins such as syndecan and also non-covalently to fibronectin fibers have been shown to bind to BMP-2 through a heparin-binding domain and regulate its bioactivity [ 37 ].

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