Bone tissue engineering—nano-glue polymer membranes for robust bone regeneration

In a new study now published on NPG Asia Materials, bioengineers report the development of a new fibrous membrane with stem cells to repair bone defects in the skulls of mice. For this, they incorporated mussel-inspired polydopamine protein as a promising compound to tether biological substances to the membrane surfaces, much like adhesive proteins in mussels. In the work, Yi Deng and a research team in the interdisciplinary departments of chemical engineering, mechanical engineering, materials technology, center for future materials and regenerative medicine in China and Australia, coated the biocompatible membranes with polydopamine nanoparticles to form many topological sites for calcium attachment and bone defect repair.

The team incubated the uncoated and coated membranes with stem cells isolated from bone marrow and implanted the membranes to regenerate skull bone defects in live mice. After a 2-month translational study, they revealed the ability of the sticky membranes to direct stem cells to produce significantly higher quantities of bone, compared with uncoated membranes. Bone defects and injuries can commonly occur at the microscopic level as congenital defects, due to accidents or as age-related degenerative disease. Most bone defects cannot be repaired spontaneously by self-healing mechanisms leading to an urgent requirement to develop robust biomaterials that facilitate bone repair in bone research and bone tissue engineering.

Bioengineers can manipulate stem cell differentiation to form mature osteoblasts via guided tissue regeneration (GTR) on surface membranes for optimized, large-scale bone regeneration. In materials science and advanced functional materials, electrospun membranes have received massive attention for such guided tissue engineering strategies due to several biocompatible advantages, including:

1. Biomimicry for stem cell attachment
2. Large surface area to facilitate cell adhesion and growth
3. The ability to form 3-D fibrous membranes and accelerate the osteogenic potential of numerous stem cell lines (from mouse, rat and human species).

Materials scientists classify synthetic GTR materials into two main categories as (1) bioabsorbable and (2) nonabsorbable materials; where nonabsorbable materials must be removed after implantation via a second surgery, causing increased healthcare expenses, while compromising newly generated tissue. In contrast, biodegradable membranes such as polyvinyl alcohol (PVA), poly (lactide-co-glycolide) (PLGA) or PCL are preferred for clinical implantations, although biological complications have seriously impeded their clinical adoption.

In the present work, therefore, Deng et al. used the bioinspired adhesive protein secreted from mussels as a 'material-independent' and facile surface coating, engineering strategy based on catechol chemistry. Polydopamine (PDA) can facilitate osteoblastic differentiation of stem cells on a variety of substrates as a nanoscale biomaterials coating, to support mesenchymal stem cell attachment and induce reprogramming of human somatic cells. Nevertheless, PDA nanolayers can easily delaminate from surfaces to induce local cell apoptosis or inflammatory reactions as adverse effects. Deng et al. implemented specific experimental steps to overcome the existing limitations and develop a new biocompatible and biodegradable membrane in the lab. The newly engineered biomaterial or fibrous membrane will provide a favorable niche to steer the fates of local stem cells to form osteoblasts for bone regeneration.

Step one: Engineering PDA-included PCL (PDA/PCL) with electrospinning

The scientists used catechol chemistry for PDA/PCL fibrous membrane fabrication and synthesis. They uniformly dispersed the PDA nanoparticles (NPs) in PCL via continuous sonication and vortexing to form the fibrous membranes via electrospinning. Deng et al. used a scanning electron microscope (SEM) and observed surface properties of the random, micron-sized fibrous network. Comparatively, the pure PCL electrospun membranes remained smooth, whereas the integration of PDA NPs made the fiber surfaces rough. The scientists confirmed the new surface chemistry using Raman spectra and X-ray photoelectron spectroscopy (XPS) analysis. The biomaterials were hydrophilic allowing protein adsorption and cell attachment. The researchers conducted contact angle measurements to verify surface wettability and improved hydrophilicity after PDA modification compared to the pure, unmodified PCL membranes.

Step two: Surface characterization studies

Since bioactive membranes can integrate with surrounding osseous tissue (bone tissue), Deng et al. assessed the formation of bone apatite layers on the surface of biomaterials immersed in simulated body fluid (SBF) solution. After 7 days of immersion, the scientists observed clustered nodular aggregates on the PDA/PCL membranes, which increased dramatically by day 14. Comparatively, the control sample of pristine unmodified PCL retained Ca-P deposits at seven days, with islands of apatite by day 14. As the PDA content increased, therefore, the amount of apatite deposited on the surface increased. Deng et al. used the material characterization data to validate enhanced in vitro bioactivity of PDA/PCL membranes compared to the pure PCL control.

Step three: Biofunctionalization studies

The scientists assessed the cytocompatibility (cell biocompatibility) of the engineered PDA/PCL membranes relative to cell adhesion, spreading, and human mesenchymal stem cell (hMSCs) proliferation. The hMSCs largely exist in bone marrow to assist tissue repair during injury. After 6 hours of cell culture, the hMSCs with round cellular shapes did not adhere well on pure PCL but expressed filopodia for membrane surface attachment on three variants of PDA/PCL membranes. Using cell counting experiments and cell viability CCK-8 assays, Deng et al. showed that the content of PDA NPs significantly affected the number of viable cells attached on the surface, and observed optimal surface properties with the 2 percent PDA/PCL group in the work.

The research team determined the optimal formula to engineer PDA/PCL membranes for guided bone tissue regeneration by determining the alkaline phosphatase (ALP) activity and calcium matrix production of hMSCs with Alizarin Red S (ARS) staining. Cell growth and osteogenic differentiation was low when the amounts of PDA NPs were either high or low, because low concentrations did not trigger cell growth, whereas high concentrations were toxic in the study.

Since investigations at the molecular level are a powerful tool in biomaterials for bioengineering, Deng et al. investigated the interplay between hMSCs and engineered membranes using molecular tools. For this, they monitored the expression of osteogenesis-related genes ALP, Runx2, Col1a1 and OPN in hMSCs cultured on the membranes. At 7 days they observed substantial levels of ALP gene expression on the 2 percent PDA/PCL sample.

By day 14, the level of Runx2 gene expressed on 1 and 2 percent PDA/PCL groups significantly outmatched the pure PCL group. However, by 21 days, the scientists did not observe a discernable difference among the four groups. They verified the observations using immunofluorescence staining and chose the 2 percent PDA/PCL membranes for optimal induction of hMSCs to differentiate into mature osteoblasts.

Step four: Translational study

Guided by the data from in vitro experiments, Deng et al. investigated the in vivo biofunctionality of the microfibrous membrane using an animal model. For this, they created critical-sized bone defects on mice skulls and placed fibrous membranes to cover the defects, followed by bone formation tests using micro-computed tomography (micro-CT), histological analyses and fluorescent labeling; four to eight weeks after implantation.

When they examined the 3-D images of the micro-CT skulls, the 2 percent PDA/PCL membrane offered the largest areas of new bone formation, with considerable expansion to the center of the bone defect. The scientists obtained a higher content of calcified matrix and bone remodeling in the 2 percent PDA/PCL membranes for phenomenal osteoconductive integration.

The hematoxylin and eosin (H&E) staining revealed fibrous tissue in the cavities of the PCL group, with comparatively visible bone restructuring in the 2 percent PDA/PCL group. The scientists also observed bone with abundant vascularization after 8 weeks post-operation in the 2 percent PDA/PCL groups. They conducted further staining with Masson, toluidine blue and immunohistochemistry (IHC) staining to identify new bone and collagen formation in depth. The combined histological data revealed that employment of PDA NPs in engineered fibrous membranes significantly boost bone regeneration, supporting the hypothesis that in vitro osteodifferentiation was also effective in vivo.

In this way, Yi Deng and co-workers bioengineered co-electrospun PDA NPs with a bioinert synthetic polymer to construct bioinspired, flexible and osteopromotive PDA/PCL fibrous membranes for bone tissue engineering applications in regenerative medicine. The amount of PDA NPs included in the composite significantly improved the chemical composition, fiber size and mechanical properties of the developed membranes. Both in vitro experiments and in vivo data validated the ability for new bone formation with 2 percent PDA/PCL constructs compared with pure PCL. The engineered PCL/PDA membranes are osteoconductive and easy to transplant with great potential for GTR applications.

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