New insights in bone tissue engineering

Rezumat:

Acest articol este o trecere in revista a  datelor din literatura de specialitate asupra celor mai recente progrese in dezvoltarea platformelor 2D si 3D pentru facilitarea repararii defectelor osoase de dimendiuni critice,cu referinta in mod special asupra Osului Flexibil,un  comus elastomeric 3D substituent al osului, integrand un hidrogel hidroxilat(pHEMA) cu 50%nHA. Modelul a fost inspirat de rolurile multiple ale nanocristalelor de hidroxiapatita ( nHA) in definirea proprietatilor structurale, mecanice si biochimice ale osului.

Abstract:

This article is a review of the literature data on the most recent  progresses in the development of scalable 2-D and 3-D scaffolds for facilitating the repair of critical-size bone defects, focusing on FlexBone, a 3-D elastomeric composite bone substitute integrating hydroxylated biocompatible pHEMA hydrogel with 50 wt% of nHA. The design was inspired by the multifaceted roles of nHA in defining the unique structural,mechanical and biochemical properties of bone.

Introduction

The  traditionally synthetic biomaterials(polymers)used in orthopedic surgery including poly (2-hydroxyethyl methacrylate) (pHEMA,terephthalate) (PET) as implant coating, polyetheretherketone (PEEK) as spacers for cervical fusion, maxillofacial defect repair, and hip prostheses[1] (PMMA) as bone cements, ultra high molecular weight polyethylene(UHMWPE) as total joint replacement components, and polysulfone (PSU) as internal fracture fixators[2]were considered bioinert.[3].Because of this feature, they lack the intrinsic ability to promote osteogenesis, thus are unable to structurally or biologically integrate with the host tissue. In an attempt to overcame this  problem,it has been modified the porosity for bending bioinert materials with  bioceramics or biodegradable polymeric components [4].

As bioactive  bone filters clinically  have been used Calcium phosphate–based bioceramics  [5] known for good biocompatibility, osteoconductivity and easy surgical handling. The main problem  with these  bone substitutes is their poor mechanical properties such as high brittleness and are often unsuitable for weight-bearing applications.[6].The solution was to integrate them  with the more compliant polymeric matrices [7].

The most investigated biodegradable synthetic  polymers which have grat potential for resorbable orthopedic implants  were: poly(glycolic acid) [8](PGA), poly(lactic-co-plycolic acid), poly(lactic acid) (PLGA),[9] polyhydroxybutyrate (PHB),[10] polycaprolactone (PCL),[11] and their copolymer  polymers used as  tissue scaffolds. The  porosity in situ generated  of degradable polymers, as a result of hydrolytic degradation, was considered  to be beneficial to tissue penetration / osteointegration.

In order to enhance  further scaffold osteoconductivity have been used biodegradable polyesters with weakly basic osteoconductive minerals such as tri-calcium phosphate (TCP) or hydroxyapatite (HA)  and for neutralizing acidic degradation products and mitigating inflammatory tissue responses.[12].

One of the most significant challenges for the clinical translation of these polymer-mineral nanocomposites for orthopedic care is achieving an  adequate structural integration between the organic matrix and the inorganic minerals represents one of the most significant challenges for the clinical translation of these polymer- mineral nanocomposites for orthopedic care.

Because most polyesters are hydrophobic in nature and exhibit an intrinsically low affinity to bioceramics,it was necessary the development of high affintity HA surface mineralization  strategies to hydrophilic hydrogels such aspoly(2-hydroxyethyl methacrylate) (pHEMA) and pHEMA-based copolymers,[13] and identification of novel HA-binding/nucleating ligands, either small molecule-based[14] or peptide-based,[15] could help address this challenge.

New achivements have been obtained in the past decade  in the design of bioactive synthetic biomaterials[16],for bone tissue engineering applications, integrin-binding peptide sequences for promoting cellular adhesion, phosphorylated ligands for promoting HA-mineralization, heparin-mimicking motifs for drug retention, and degradative enzyme substrate sequences have all been incorporated into multi-modality synthetic scaffold designs.,[17]such as the design of self-assembling peptideamphiphile (PA) gels by Stupp and coworkers for simultaneous presentation of cell adhesion peptide sequences, HA-mineral-nucleating sites, reversible crosslinking sites, and other therapeutic agents all within a single PA molecule that self-assembles and dissembles in response to environmental perturbations[18].

The disadvantage of  these unique PA gels include their relatively high manufacturing cost and low mechanical modulus which could limit their use to treatment of small non-weight bearing skeletal lesions.

Hubbel and coworkers introduced another innovative concept to induce scaffold degradation by using peptide substrates of the degradative enzymes matrix metalloproteinases (MMPs) as the chemical crosslinker of a non-fouling crosslinked hydrogel system[19] Due to  the elevated expression of some MMPs within both degenerative bony defects and arthritic knee joints, such a hydrogel system could be useful for bone and cartilage repair as the in situ increase of scaffold porosity in response to tissue microenvironment-specific enzymatic degradation could promote cellular infiltration and matrix deposition. It is not a trivial task the selection of MMP substrates with proper degradation kinetics matching with those of the matrix deposition rate.

The problem is that despite the many exciting orthopedic biomaterials emerging in the literature, successful clinical translations are rare because of the difficulty in accomplishing the functional sophistication of viable synthetic bone substitutes  (e.g. physical properties enabling easy surgical handling and stable graft fixation, structural and biomechanical properties facilitating its osteointegration, biocompatibility ensuring long-term safety) within an easy-to-fabricate biomaterial that can be reproducibly manufactured at low cost.

The bone tissue composition

Bone ,from a material’s perspective, is an organic-inorganic composite comprising two major structural components that are hierarchically organized across various length scales: the calcium apatite crystals (primarily as substituted nanocrystalline hydroxyapatite, nHA, but also as crystalline precursors in lower quantities) and the type I collagen matrix[20].

The mechanical properties of bone is influenced by the  quantity and quality of the hard calcium apatite crystals (crystal size, maturity and structural integration with the collagen matrices)[21] For example, the bending and compression strength of bone is known to positively correlate to bone mineral content.[22]In addition, bone minerals also support bone cell attachment, serve as an important reservoir of calcium and phosphate ions, and help retain the secreted factors that are indispensable in regulating the biochemical microenvironment of the bony tissue.

Therefore, HA has long been recognized as an important design element for tissue-engineered bone substitutes.[23] The inspiration to  use of bioceramic scaffold,[24] or polymer-bioceramics composite scaffolds came from intrinsic affinity of the dynamic apatite crystal surface for many acidic non-collagenous proteins widely found in calcified tissues[21]  to retain and deliver recombinant proteins for therapeutic use. Bone tissue engineering have explored opportunity of using HA more as a way to enhance the mechanical strength than as a tool to mediate the biochemical properties of the scaffold.[25]

The  potential of the large surface areas provided by nHA as opposed to micrometer-sized HA for more efficient therapeutics delivery (e.g. higher retention capacity, more sustained release) has not been exploited to the fullest extent in the design of synthetic bone substitutes.

It is well known that type I collagen matrix of bone serves as a compliant template for the structural integration of the calcium apatite crystals, and, along with the mineral component, is responsible for defining the 3-dimensional structure as well as the strong, tough, yet compliant mechanical properties of bone.[26] It also interacts with many non-collagenous proteins and mediates cellular adhesion and functions.

Also,the Gly-Pro-Hyp (Hyp: hydroxyproline) triplet repeats of type I collagen may also play an important role in template-driven biomineralization. Recent discovery of novel HAbinding oligopeptides using the combinatorial phage display technique reveals a [Pro-(OH)-X] tripeptide pattern (OH: hydroxylated amino acid residues (Ser, Thr, Tyr); X: any amino acid) among the dominant HA-binding motifs,resembling  that of the type I collagen, underscoring the importance of hydroxylated residues in directing ligand-mineral interactions on a molecular level.

Also,these oligopeptides were shown to template the nucleation and growth of HA in vitro and may be useful in the design of synthetic polymer scaffolds, enabling template-driven mineralization of HA or the preparation of bulk organic-inorganic bone-like composites with improved interfacial binding affinity.The  polymeric hydrogels displaying hydroxylated (e.g. pHEMA) and acidic residues could be used to template the surface mineralization of HA with excellent interfacial adhesion strength, further supporting the favorable interaction between the hydroxyls and the calcium ions.

Also,the strategy of modifying the surface of polymers or metallic substrates with hydroxylated or anionic coatings has also been pursued to facilitate the nucleation and growth of calcium apatite.[27]

Sinthetic bone scaffolds

There are  a great number  of proteins are known to play roles in the biological healing of bone,including in cellular recruitment and initiating the inflammation / bone remodeling cascades.[28].  Most scaffold-assisted bone repair would require the supplement of exogenous therapeutic agents such as osteogenic growth factors to augment the biological performance of biomaterial scaffolds. The most commonly used clinically  therapeutic agents to enhance bone repair are the Food and Drug Administration (FDA)-approved osteogenic growth factor bone morphogenetic protein -2(BMP-2) and BMP-7.[29]

Also, vascular endothelial growth factor (VEGF),[30]receptor activator of nuclear factor kappa-Β ligand (RANKL) and transforming growth factor β (TGFβ)[30] are also used to modulate the graft vascularization, osteoinegration and remodeling. BMP-2/7, is another recombinant factor that has gained quick attention within the bone tissue engineering community which is  a protein heterodimer of BMP-2 and BMP-7 that is more potent in inducing osteogenic differentiation of pluripotent cells in vitro than either homodimer alone.[31] Its higher potency has been attributed to its decreased sensitivity to BMP inhibitors.

In particular, BMP-2/7 was found to have decreased sensitivity to Noggin, a BMP antagonist that is secreted from mesenchymal cells in response to BMPs to help control the rate of cellular differentiation. It is known that Noggin binds BMP, inhibiting BMP cell surface receptor binding; however, Noggin’s binding affinity is lowered in the BMP-2/7 heterodimer. The high potency of BMP-2/7 has important clinical implications, including more cost-effective low-dose treatment with reduced systemic side-effects. There a a great  number of laboratories, which  have exploited the use of BMP-2/7 for augmenting scaffold-assisted repair of bone in vivo.[32]

Most recent improvements in biomaterials’ therapeutic delivery characteristics for bone repair were accomplished by incorporating nHA in polymeric hydrogel matrices,  or by combining thin fibrous film-based physical barriers with 3-dimensional hydrogels[33] to enable the retention and release of growth factors in a more localized and sustained manner.Another innovative approach is physical entrapment of therapeutic agents within MMP-degradable poly(ethylene glycol) (PEG)-based hydrogels that has been established for therapeutic delivery by Hubbell and coworkers.[34]

The use of electrostatic interactions in hydrogels is another, more established, method for therapeutic delivery,for example, gelatin hydrogels can be basic and positively charged, or acidic and negatively charged depending on how the gelatin is extracted from collagen, thus can be designed to interact with a wide range of charged therapeutics.Also an effective method proven was  Hyaluronic acid incorporation into hydrogels  for not only therapeutic retention of proteins, such as BMP-2, but also for cellular adhesion, migration and proliferation, as well as binding to collagen and fibrin[35].

Also,sulfates have  been exploited for therapeutic retention. Hydrogels that incorporate negatively charged heparin sulfate can easily interact with many positively charged proteins, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and BMP-2[36].

In comparison with the latter strategies, the incorporation of nHA has the added advantages of being able to retain and enrich endogenously secreted factors due to its intrinsic affinity to the factors residing in the bony tissue environment and its large surface area available for absorption.

Complimentary synthetic scaffolds

2-Dimensional vs. 3-Dimensional

Biomaterials for bone repair can be enhanced by the addition of exogenous therapeutics,but, they can also benefit from exogenous cells (e.g. mesenchymal stem cells,hematopoietic cells, osteoblasts) pre-seeded on the biomaterial scaffolds.

The cellular attachement  of both endogenous cells and pre-seed exogenous cells has been improved using  collagenous protein mimetics of extracellular matrix components biomaterials with integrin binding peptides such as RGD.[37]

For bone repair are attracted all materials possessing the ability to support cellular encapsulation (e.g. 3-D constructs) or surface attachment (e.g. 2-D films or fiber meshes) and that favor specific stem cell differentiation pathways, in addition to the ability to act as a therapeutic delivery vehicle.

Towards this goal have ben exploited , both  3-D and 2-D constructs ,104-106 although greater focus has been placed on engineering 3-D scaffolds. A focus has been centred among the 2-D platforms explored, on supporting cellular attachment and differentiation (due to the relative ease of cell attachment on 2-D compared to 3-D scaffolds), as well as delivery of osteogenic factors in culture.[38]

The unique handling characteristics of 2-D scaffolds could enable versatile in vivo uses, including as a stand-alone graft overlying a fracture, a filler being press-fit into an area of small bony defect, or a synthetic membrane wrapped around a 3-D bone graft.

The combination of 2-D and 3-D constructs has recently been explored by Robert Guldberg and colleagues to achieve spatio-temporal control of growth factor release profiles,[38]  and by others to create hierarchical composites[39].

Starting from a biomimetic perspective, 2-D scaffolds that can be wrapped around a 3-D bone scaffold, if engineered properly, can recapitulate some of the important functions of periosteal tissues surrounding long bone in harboring stem cell and directing their differentiation upon injury.

BMP2 and BMP7 are 3-D graft components-  BMP-2 is known to play a critical role in initiating early bone healing while BMP-7 may be more suitably used for stimulating later stages of bone healing[40] One could envision using a 3-D scaffold designed for slower release of BMP-7 in combination with a 2-D scaffold designed for faster release of BMP-2.

Alternatively, different cell types could be seeded on each construct, such as seeding bone marrow-derived stem cells on the 2-D construct to more efficiently initiate graft healing while seeding hematopoietic stem cells on the 3-D construct to promote the vascularization of the graft for better tissue incorporation. In cases where the 3-D grafts (i.e. allografts) cannot readily support the seeding of exogenous cells, a cell-laden 2-D construct could be readily wrapped around the 3-D graft.

Electrosppining is a common method for preparing a 2-D fibrous mesh scaffold with controlled fiber dimension, mesh thickness and porosity is by electrospinning. When the polymer fibers are properly chosen, they can be subjected to further chemical modifications to render specific biological properties and/or mechanically strengthened to improve their handling characteristics.[41]

Conclusion

The progress of future research in orthopedics  will bring new strategies to assure a normal bone formation cascade as well as bone disease of different origins to be  treated with novel bone regeneration protocols.

References:

1. Eschbach, L. Nonresorbable polymers in bone surgery. Injury 31 Suppl 4, 22, 2000.
2. Wang, M. Developing bioactive composite materials for tissue replacement. Biomaterials 24, 2133, 2003.
3. Mano, J.F., Sousa, R.A., Boesel, L.F., Neves, N.M., and Reis, R.L. Bloinert, biodegradable and injectable polymeric matrix composites for hard tissue replacement: state of the art and recent developments. Composites Science and Technology 64, 789, 2004.
4. Aparecida, A.H., Guastaldi, A.C., and Fook, M.V.L. Development of Ultra High Molecular Weight Polyethylene (UHMWPE) Porous Supports for Use as Biomaterial in Osseous Replacement and Regeneration. Polimeros 18, 277, 2008.
5. Nandi, S.K., Roy, S., Mukherjee, P., Kundu, B., De, D.K., and Basu, D. Orthopaedic applications of bone graft & graft substitutes: a review. Indian Journal of Medical Research 132, 15, 2010.
6. Tanner, K.E. Bioactive composites for bone tissue engineering. Proceedings of the Institution of Mechanical Engineers Part H-Journal of Engineering in Medicine 224, 1359, 2010.
7. Wang, M., and Bonfield, W. Chemically coupled hydroxyapatite-polyethylene composites: structure and properties. Biomaterials 22, 1311, 2001.
8. Zhang, R.Y., and Ma, P.X. Poly(alpha-hydroxyl acids) hydroxyapatite porous composites for bone-tissue engineering. I. Preparation and morphology. J Biomed Mater Res 44, 446, 1999.
9. Ishaug, S.L., Crane, G.M., Miller, M.J., Yasko, A.W., Yaszemski, M.J., and Mikos, A.G. Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds. J Biomed Mater Res 36, 17, 1997.
10. Wang, Y.W., Wu, Q.O., and Chen, G.Q. Attachment, proliferation and differentiation of osteoblasts on random biopolyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) scaffolds. Biomaterials 25, 669, 2004.
11. Yoshimoto, H., Shin, Y.M., Terai, H., and Vacanti, J.P. A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials 24, 2077, 2003.
12. Liao, S., Watari, F., Zhu, Y., Uo, M., Akasaka, T., Wang, W., Xu, G., and Cui, F. The degradation of the three layered nano-carbonated hydroxyapatite/collagen/PLGA composite membrane in vitro. Dental Materials 23, 1120, 2007.
13. Song, J., Malathong, V., and Bertozzi, C.R. Mineralization of synthetic polymer scaffolds: A bottom-up approach for the development of artificial bone. Journal of the American Chemical Society 127, 3366, 2005.
14. Licata, A.A. Discovery, clinical development, and therapeutic uses of bisphosphonates. Ann Pharmacother 39, 668, 2005.
15. Chung, W.J., Kwon, K.Y., Song, J., and Lee, S.W. Evolutionary Screening of Collagen-like Peptides That Nucleate Hydroxyapatite Crystals. Langmuir, dx.doi.org/10.1021/la104757g, 2011.
16. Bonzani, I.C., George, J.H., and Stevens, M.M. Novel materials for bone and cartilage regeneration. Current Opinion in Chemical Biology 10, 568, 2006.
17. Patterson, J., and Hubbell, J.A. Enhanced proteolytic degradation of molecularly engineered PEG hydrogels in response to MMP-1 and MMP-2. Biomaterials 31, 7836, 2010.
18. Hartgerink, J.D., Beniash, E., and Stupp, S.I. Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 294, 1684, 2001.
19. Lutolf, M.R., Weber, F.E., Schmoekel, H.G., Schense, J.C., Kohler, T., Muller, R., and Hubbell, J.A. Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nature Biotechnology 21, 513, 2003.
20. Weiner, S., Traub, W., and Wagner, H.D. Lamellar bone: Structure-function relations. Journal of Structural Biology 126, 241, 1999.
21. Tong, W., Glimcher, M.J., Katz, J.L., Kuhn, L., and Eppell, S.J. Size and shape of mineralites in young bovine bone measured by atomic force microscopy. Calcified Tissue International 72, 592, 2003.
22. Follet, H., Boivin, G., Rumelhart, C., and Meunier, P.J. The degree of mineralization is a determinant of bone strength: a study on human calcanei. Bone 34, 783, 2004.
23. El-Ghannam, A. Bone reconstruction: from bioceramics to tissue engineering. Expert Review of Medical Devices 2, 87, 2005
24. Le Nihouannen, D., Hacking, S.A., Gbureck, U., Komarova, S.V., and Barralet, J.E. The use of RANKL-coated brushite cement to stimulate bone remodelling. Biomaterials 29, 3253, 2008.
25. Stevens, M.M. Biomaterials for bone tissue engineering. Materials Today 11, 18, 2008.
26. Scharnweber, D., Born, R., Flade, K., Roessler, S., Stoelzel, M., and Worch, H. Mineralization behaviour of collagen type I immobilized on different substrates. Biomaterials 25, 2371, 2004.
27. Zhang, H.L., Simpson, D., Kumar, S., and Smart, R.S.C. Interaction of hydroxylated PACVD silica coatings on titanium with simulated body fluid. Colloids and Surfaces a-Physicochemical and Engineering Aspects 291, 128, 2006.
28. Einhorn, T.A. The cell and molecular biology of fracture healing. Clin Orthop Relat Res, S7, 1998.
29. Gautschi, O.P., Frey, S.P., and Zellweger, R. Bone morphogenetic proteins in clinical applications. ANZ J Surg 77, 626, 2007.
30. Patel, Z.S., Young, S., Tabata, Y., Jansen, J.A., Wong, M.E.K., and Mikos, A.G. Dual delivery of an angiogenic and an osteogenic growth factor for bone regeneration in a critical size defect model. Bone 43, 931, 200.
31. Zheng, Y.N., Wu, G., Zhao, J., Wang, L.H., Sun, P., and Gu, Z.Y. rhBMP2/7 Heterodimer: An Osteoblastogenesis Inducer of Not Higher Potency but Lower Effective Concentration Compared with rhBMP2 and rhBMP7 Homodimers. Tissue Eng Part A 16, 879, 2010.
32. Koh, J.T., Zhao, Z., Wang, Z., Lewis, I.S., Krebsbach, P.H., and Franceschi, R.T. Combinatorial gene therapy with BMP2/7 enhances cranial bone regeneration. Journal of Dental Research 87, 845, 2008.
33. Kolambkar, Y.M., Boerckel, J.D., Dupont, K.M., Bajin, M., Huebsch, N., Mooney, D.J., Hutmacher, D.W., and Guldberg, R.E. Spatiotemporal delivery of bone morphogenetic protein enhances functional repair of segmental bone defects. Bone, 2011.
34. Lutolf, M.P., Weber, F.E., Schmoekel, H.G., Schense, J.C., Kohler, T., Muller, R., and Hubbell, J.A. Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nat Biotechnol 21, 513, 2003.
35.  Kim, J., Kim, I.S., Cho, T.H., Lee, K.B., Hwang, S.J., Tae, G., Noh, I., Lee, S.H., Park, Y., and Sun, K. Bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and human mesenchymal stem cells. Biomaterials 28, 1830, 2007.
36. Benoit, D.S.W., and Anseth, K.S. Heparin functionalized PEG gels that modulate protein adsorption for hMSC adhesion and differentiation. Acta Biomater 1, 461, 2005.
37. Holmes, T.C. Novel peptide-based biomaterial scaffolds for tissue engineering. Trends in Biotechnology 20, 16, 2002.
38. Kolambkar, Y.M., Dupont, K.M., Boerckel, J.D., Huebsch, N., Mooney, D.J., Hutmacher, D.W., and Guldberg, R.E. An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects. Biomaterials 32, 65, 2011.
39. Yeo, M., Lee, H., and Kim, G. Three-Dimensional Hierarchical Composite Scaffolds Consisting of Polycaprolactone, beta-Tricalcium Phosphate, and Collagen Nanofibers: Fabrication, Physical Properties, and In Vitro Cell Activity for Bone Tissue Regeneration. Biomacromolecules 12, 502, 2011.
40. Onishi, T., Ishidou, Y., Nagamine, T., Yone, K., Imamura, T., Kato, M., Sampath, T.K., ten Dijke, P., and Sakou, T. Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type II receptor during fracture healing in rats. Bone 22, 605, 1998.
41. Pham, Q.P., Sharma, U., and Mikos, A.G. Electrospinning of polymeric nanofibers for tissue engineering applications: A review. Tissue Engineering 12, 1197, 2006.

Gabriel Ovidiu Dinu

Floreasca Universitary Emergency Hospital