Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Author: Tilda Barliya PhD

Annual treatment costs for musculoskeletal diseases in the US are roughly 7.7% (~ $849 billion) of total gross domestic product. Such disorders are the main cause of physical disability in US (I). The challenges of drug delivery for bone regeneration and reconstruction has been previously reported here by Dr. Aviral Vatsa (I-IV), herein, we will discussed the different needs for bone regeneration and the potential use if nanotechnology.

Bone regeneration is a complex, well-orchestrated physiological process of bone formation, which can be seen during normal fracture healing, and is involved in continuous remodelling throughout adult life. However, there are complex clinical conditions in which bone regeneration is required in large quantity, such as for skeletal reconstruction of large bone defects created by trauma, infection, tumour resection and skeletal abnormalities, or cases in which the regenerative process is compromised, including avascular necrosis, atrophic non-unions and osteoporosis (1,2).

Regenerative medicine offers a way to improve  ‘local’ strategies in terms of tissue engineering and gene therapy, or even ‘systemic’ enhancement of bone repair. To make regenerative medicine successful, three elements are required: stem cells, scaffolds, and growth factors (3).

Bones

Bone is a tough supporting tissue and functions in both movement and the maintenance of postural stability by working cooperatively with muscles as well as play a role in calcium metabolism. Despite its hard structure it exist in a dynamic turnover known as bone remodeling. There are two types of bone structures that naturally remodel during the a year:

• cortical bone (~3%/year)
• cancellous bone (~30%/year)

At the remodeling sites, osteoblasts produce new bone, while osteoclasts resorb existing bone. Each cell type seems to be regulated by a variety of hormones and by local factors. If the balance between bone formation and resorption is lost by uncontrolled production of these regulators, the bone structure will be damaged, and the subject would be susceptible to osteoporosis and osteopetrosis (2).

Current Clinical approaches:

Standard approaches widely used in clinical practice to stimulate or augment bone regeneration include distraction osteogenesis and bone transport.

As well as the use of a number of different bone-grafting methods, such as (1):

• Autologous bone grafts – considered as the ‘gold standard‘ bone-grafting material, as it combines all properties required in a bone-graft material: osteoinduction (bone morphogenetic proteins (BMPs) and other growth factors), osteogenesis (osteoprogenitor cells) and osteoconduction (scaffold)
• Allografts – obtained from human cadavers or living donors, which bypasses the problems associated with harvesting and quantity of graft material. Allogeneic bone is available in many preparations, including demineralised bone matrix (DBM), morcellised and cancellous chips, corticocancellous and cortical grafts, and osteochondral and whole-bone segments, depending on the recipient site requirements.
• Bone-graft substitutes or growth factors – developed as alternatives to autologous or allogeneic bone grafts. They consist of scaffolds made of synthetic or natural biomaterials that promote the migration, proliferation and differentiation of bone cells for bone regeneration. Commonly performed surgical procedure to augment bone regeneration in a variety of orthopaedic and maxillofacial procedures.

The Masquelet technique is a two-step procedure for bone regeneration and reconstruction of long-bone defects. It is based on the concept of a “biological” membrane, which is induced after application of a cement spacer at the first stage and acts as a ‘chamber’ for the insertion of non-vascularised autograft at the second stage (2, 4).

There are  non-invasive methods of biophysical stimulation, such as low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic fields (PEMF) (1).

Limitations of Current approaches: Most of the current strategies for bone regeneration exhibit relatively satisfactory results. However, there are associated drawbacks and limitations to their use and availability, and even controversial reports about their efficacy and cost-effectiveness.

New Approaches:

New methods for studying this process, such as quantitative three-dimensional microcomputed tomography analyses, finite element modelling, and nanotechnology have been developed to further evaluate the mechanical properties of bone regenerate at the microscopic level. Here are some examples of the latest developments as reviewed by Dimitriou R at el (1).

BMPs and growth factors – They induce the mitogenesis of mesenchymal stem cells (MSCs) and other osteoprogenitors, and their differentiation towards osteoblasts. BMP-2 and BMP-7 have been licensed for clinical use since 2002 and 2001 respectively (5). These two molecules have been used in a variety of clinical conditions including non-union, open fractures, joint fusions, aseptic bone necrosis and critical bone defects. Platelet-derived growth factor (PDFG), transforming growth factor-β (TGF-b), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) have been also implicated in bone regeneration, with different functions in terms of cell proliferation, chemotaxis and angiogenesis. One current approach to enhance bone regeneration and soft-tissue healing by is local application of growth factors is the use of platelet-rich plasma alongside the autograph. BMPs are also being used in bone-tissue engineering.

MSCs – The current approach of delivering osteogenic cells directly to the regeneration site includes use of bone-marrow aspirate from the iliac crest, which also contains growth factors. It is a minimally invasive procedure to enhance bone repair, and produces satisfactory results (1). Overall, however, there are significant ongoing issues with quality control with respect to delivering the requisite number of MSCs/osteoprogenitors to effect adequate repair responses. Issues of quantity and alternative sources of MSCs are being extensively investigated. Novel approaches in terms of cell harvesting, in vitro expansion and subsequent implantation are promising.

Scaffolds and Bone substitutes – synthetic bone substitutes and biomaterials are already widely used in clinical practice for osteoconduction. DBM (Demineralized bone matrix)  and collagen are biomaterials, used mainly as bone-graft extenders, as they provide minimal structural support. A large number of synthetic bone substitutes are currently available, such as HA, β-TCP and calcium-phosphate cements, and glass ceramics. These are being used as adjuncts or alternatives to autologous bone grafts. Especially for regeneration of large bone defects, where the requirements for grafting material are substantial, these synthetics can be used in combination with autologous bone graft, growth factors or cells (6). Improved biodegradable and bioactive three-dimensional porous scaffolds are being investigated, as well as novel approaches using nanotechnology, such as magnetic biohybrid porous scaffolds acting as a crosslinking agent for collagen for bone regeneration guided by an external magnetic field or injectable scaffolds for easier application.

Tissue Engineering – The tissue-engineering approach is a promising strategy added in the field of bone regenerative medicine, which aims to generate new, cell-driven, functional tissues, rather than just to implant non-living scaffolds. In essence, bone-tissue engineering combines progenitor cells, such as MSCs (native or expanded) or mature cells (for osteogenesis) seeded in biocompatible scaffolds and ideally in three-dimensional tissue-like structures (for osteoconduction and vascular ingrowth), with appropriate growth factors (for osteoinduction), in order to generate and maintain bone (7). Bone-tissue engineering is in its early stages, and there are many issues of efficacy, safety and cost to be addressed before general clinical application can be achieved.

Gene Therapy – This involves the transfer of genetic material into the genome of the target cell, allowing expression of bioactive factors from the cells themselves for a prolonged time. Gene transfer can be performed using a viral (transfection) or a non-viral (transduction) vector, and by either an in vivo or ex vivo gene-transfer strategy. There are issues of cost, efficacy and biological safety that need to be answered.

Nanotechnology and Bone Regeneration

Nanotechnology has been greatly utilized for bone tissue engineering strategies. It has been employed to overcome some of the current limitations associated with bone regeneration methods including insufficient mechanical strength of scaffold materials, ineffective cell growth and osteogenic differentiation at the defect site, as well as unstable and insufficient production of growth factors to stimulate bone cell growth (8,9).

To mimic the natural bone nanocomposite architecture, novel biomaterials and nanofabrication techniques are currently being employed and many different nanostructures have already been designed and tested. Electrospinning has been extensively applied to create bone nanofiber scaffolds and biomaterials typically used for this purpose, including synthetic organic polymers such as PCL, PLGA, PLLA, Chitosan, and silk fibroin.

Among the materials used for bone-reconstruction, PLLA is a biocompatible polymer with the advantage of being highly. biodegradable. For this reason, PLLA have received the approval of the Food and Drug Administration (FDA) to be use in bone reconstructive surgery (10).

PLLA nanofibers are often functionalized to improve their biological performance with peptides such as RGD (Arg-Gly-Asp); with osteogenic molecules such as hydroxyapatite; or with proteins such as collagen and the growth factor bone morphogenic protein 2 (BMP-2). It was found that direct incorporation of BMP-2 into PLLA nanofibers enhances the osteoinductivity of the scaffolds.

Current orthopedic implants fail in an appropriate osteo-integration limiting implant lifespan. Titanium, as a biocompatible material, has been used to enhance implant incorporation in bone for dental, craniofacial, and orthopedic applications. Studies have demonstrated that nanoporous titanium dioxide (TiO2) surface modification alters nanoscale topography improving soft tissue attachment on titanium implants surface (11). For example, the uses of nanoporous TiO2 surface-modified implants, in a human dental clinical study, showed that TiO2 thin film increased adherence in early healing of the human oral mucosa and reduced marginal bone resorption (11).

Another example are rosette nanotubes. Bioactive helical rosette nanotubes are self-assembled nanomaterials, formed in water from synthetic DNA base analogs that mimic the helical nanostructure of collagen in bone. This technology has been used to create a biomimetic nanocomposite combined with nanocrystalline hydroxyapatite, and biocompatible hydrogels which increased osteoblast adhesion.

Carbon nanotubes (CNTs) are other suitable scaffold materials that have proved to support osteoblast proliferation. CNTs possess exceptional mechanical, thermal, and electrical properties, facilitating their use as reinforcements or, in combination with other biomaterials, to improve and to support bone growth.

Nanotechnology and clinical trials

Clinical therapies implying the use of nanotechnology in bone regeneration are still in the beginning stages.

BDSint –  Recently, the bone healing ability of a nanocomposite (DBSint®), approved for clinical use, constituted by biomimetic nanostructured Mg-hydroxyapatite and human demineralized bone matrix has been investigated.  The clinical-radiographic and histomorphometry study in subjects undergoing high tibial osteotomy, demonstrated that these nanocomposites are safe and effective. Yet the long term outcome is still to be defined (8, 12).

BioOsss and BioGides –  Schwarz et al. undertook a four-year study of patients treated of moderate intrabony peri-implantitis defects using either a nanocrystalline hydroxyapatite or a natural bone mineral (BioOsss spongiosa granules) in combination with a collagen membrane (BioGides) and found bone reconstruction (8, 13).

Here are some of the ongoing clinical trials for use of nanotechnology in bone regeneration (Perán M et al (8)):

NCT00729716 – Comparison of BioCart™II With Microfracture for Treatment of Cartilage Defects of the Femoral Condyle BioCart™II scaffold Cartilage ————Phase 2.

NCT01183637  – Evaluation of “Kensey Nash Corp” an Acellular Osteochondral Graft for Cartilage Lesions Pilot Trial (EAGLE Pilot) bioresorbable scaffold Bone/ Cartilage————-Phase 2

NCT01218945 –  Development of Bone Grafts Using Adipose-Derived Stem Cells and Different Scaffolds Bone scaffold Bone——– recruiting participants

NCT01435434 – Mononucleotide Autologous Stem Cells and Demineralized Bone Matrix in the Treatment of Non-Union/Delayed Fractures Ignite®ICS injectable scaffold Bone——————Not yet recruiting

Summary:

The advantages of nanomaterials as therapeutic and diagnostic tools are vast, due to design flexibility, small sizes, large surface-to-volume ratio, and ease of surface modification.  The potential of these bio-devices has shown promising results in vitro, and some of them have also been successfully tested in vivo with animal models. Nevertheless, the gap between laboratory and medical application of these nanotechnological advances is still wide (8).

Although some successful devises have already being tested in clinical trials and the data produced by these studies is highly encouraging, the safety of nanomedicine is not yet fully defined and more clinical studies still need to be conducted to translate nanotechnological devices to the clinic.

Reference:

1. Dimitriou R, Jones E, McGonagle D and Giannoudis P.V. Bone regeneration: current concepts and future directions. BMC Medicine 2011, 9:66. http://www.biomedcentral.com/1741-7015/9/66

2. Jimi E.,  Hirata S., Osawa K.,  Terashita M., Kitamura C.,  and Fukushima H. The Current and Future Therapies of Bone Regeneration to Repair Bone Defects. International Journal of Dentistry Volume 2012 (2012), Article ID 148261. doi:10.1155/2012/148261. http://www.hindawi.com/journals/ijd/2012/148261/

3. G. C. Gurtner, M. J. Callaghan, and M. T. Longaker, “Progress and potential for regenerative medicine,” Annual Review of Medicine, vol. 58, pp. 299–312, 2007. http://www.ncbi.nlm.nih.gov/pubmed/17076602

4. Masquelet AC, Begue T: The concept of induced membrane for reconstruction of long bone defects. Orthop Clin North Am 2010, 41(1):27-37. http://www.ncbi.nlm.nih.gov/pubmed/19931050

5. Food and Drug Administration: Medical devices. [http:/ / www.fda.gov/ MedicalDevices/ ProductsandMedicalProcedures/ DeviceApprovalsandClearances/ Recently-ApprovedDevices/ default.htm

6. Giannoudis PV, Dinopoulos H, Tsiridis E: Bone substitutes: an update. Injury 2005, 36(Suppl 3):S20-27. http://www.ncbi.nlm.nih.gov/pubmed/16188545

7. Jones E, English A, Churchman SM, Kouroupis D, Boxall SA, Kinsey S, Giannoudis PG, Emery P, McGonagle D: Large-scale extraction and characterization of CD271+ multipotential stromal cells from trabecular bone in health and osteoarthritis: implications for bone regeneration strategies based on uncultured or minimally cultured multipotential stromal cells. Arthritis Rheum 2010, 62(7):1944-1954.  http://onlinelibrary.wiley.com/doi/10.1002/art.27451/abstract;jsessionid=4573A69E4561194C83A97EC302CD20CB.d04t02

8. Perán M., García MA., Lopez-Ruiz E., Jiménez G and Marchal JA. How Can Nanotechnology Help to Repair the Body?Advances in Cardiac, Skin, Bone, Cartilage and Nerve Tissue Regeneration. Materials 2013, 6, 1333-1359; doi:10.3390/ma6041333 http://www.mdpi.com/1996-1944/6/4/1333

9. Kim K and Fisher JP. Nanoparticle technology in bone tissue engineering. J Drug Target. 2007 May;15(4):241-52.  http://www.ncbi.nlm.nih.gov/pubmed/17487693

10.  Schofer, M.D.; Roessler, P.P.; Schaefer, J.; Theisen, C.; Schlimme, S.; Heverhagen, J.T.; Voelker, M.; Dersch, R.; Agarwal, S.; Fuchs-Winkelmann, S.; Paletta, J.R. Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects. PLoS One 2011, 6, e25462. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182232/

11.  Wennerberg, A.; Frojd, V.; Olsson, M.; Nannmark, U.; Emanuelsson, L.; Johansson, P.; Josefsson, Y.; Kangasniemi, I.; Peltola, T.; Tirri, T.; et al. Nanoporous TiO(2) thin film on titanium oral implants for enhanced human soft tissue adhesion: a light and electron microscopy study. Clin. Implant. Dent. Relat. Res. 2011, 13, 184–196. http://www.ncbi.nlm.nih.gov/pubmed/19681943

12. Dallari, D.; Savarino, L.; Albisinni, U.; Fornasari, P.; Ferruzzi, A.; Baldini, N.; Giannini, S. A prospective, randomised, controlled trial using a Mg-hydroxyapatite-demineralized bone matrix nanocomposite in tibial osteotomy. Biomaterials 2012, 33, 72–79. http://www.ncbi.nlm.nih.gov/pubmed/21955688

13. Schwarz, F.; Sahm, N.; Bieling, K.; Becker, J. Surgical regenerative treatment of peri-implantitis lesions using a nanocrystalline hydroxyapatite or a natural bone mineral in combination with a collagen membrane: a four-year clinical follow-up report. J. Clin. Periodontol. 2009, 36, 807–814.  http://www.ncbi.nlm.nih.gov/pubmed/19637997

Other articles from our Open Access Journal

I. By: Aviral Vatsa PhD MBBS. Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part I. https://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

II. By: Aviral Vatsa PhD MBBS. Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part II. https://pharmaceuticalintelligence.com/2012/09/30/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-ii/

III. By: Aviral Vatsa PhD MBBS. Osteocytes: A Special Issue in Bone.  https://pharmaceuticalintelligence.com/2013/02/06/osteocytes-a-special-issue-in-bone/

IV. By: Aviral Vatsa PhD MBBS. Bone remodelling in a nutshell. https://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

V. By: Ritu Saxena PhD. Dual protection of bone by Sema3a. https://pharmaceuticalintelligence.com/2012/05/10/dual-protection-of-bone-by-sema3a-2/