A New Age in Regenerative Medicine

How we can optimize nano-sized particles/materials to heal patients

Introduction

Being a very passionate football fan, I enjoy watching the many NFL games every Sunday. However, there has never seemed to have been a week where at least 1 person did not get injured. Whether it be a rib injury, torn ACL/MCL, torn hamstring, bones broken, etc., all the players usually don’t get another chance to play that season since they have to heal and recover— extremely disheartening to see.

Not just in professional sports, but people in general suffer from many injuries that are life-threatening or take much too long to heal, putting a halt on their lives. From skin burns, to replacement nerves, we need new ways to tackle the long overdue problem of regenerative medicine.

Essentially, we need to be able to “fix” patients in a cost-effective, safe, efficient, and timely manner. Current methods (ie. surgery, medicines, organ transplants, etc.) don’t fulfill all 4 aspects, making it that much more of a barrier for these health crises. Well, if that’s the case, what can be used?

Enter nanotechnology. If you haven’t already, I suggest you check out this article, where I explain what this technology is, and how it works. Good to go? Then let's get started.

Today, I want to talk to you about how we can use nanomaterials to regenerate skin wounds, reconstruct bones, repair nerve damage, and finally heal cardiac tissue. It's going to be very “sciency” so feel free to jump around from section to section depending on what interest you, and if you so please, head to the bottom and just read a quick summary!

Terms to know:

In vitro: Work that’s performed outside of a living organism. This can include studying cells in culture or methods of testing the antibiotic sensitivity of bacteria.

In vivo: When research or work is done with or within an entire, living organism

Skin Regeneration

Quick note: Learn about electrospinning here to understand some of the processes

The primary function of the skin is to act as a barrier. Any related problem to the skin such as burns, chronic wound, ulcers or accidents can cause serious health complications. The apparently simple structure of the skin, consisting of two layers, the epidermis and the dermis, and its easy target localization, has encouraged the search for therapeutic alternatives. In this regard, nanotech emerges as a promising hope to improve wound healing and skin restoration.

Skin tissue engineering is based in the creation of “scaffolds” that must share the followed minimal characteristics: biocompatibility, support for cell attachment and proliferation, and to imitate the extracellular structure as closely as possible.

One of the main difficulties found in the application of this artificial skin is the problems related to adhesion and integration of the scaffolds to the topography of the wound, while maintaining physical and mechanical properties.

Electrospun nano-fibrous scaffolds have been created to mimic the three-dimensional fiber network of the collagen fibrous structure found in the skin. They are composed of collagen fibers that are formed hierarchically by nanometer-scale multi-fibrils and have been proved to support cell adhesion, proliferation, and differentiation mimicking the fibrous architecture of the extracellular structure (ie. very good).

In addition, the electrospinning technique allows for control over the desired pore diameter, distribution, total volume, total area, and, consequently, the final porosity of the structure.

An in vivo study has shown the benefits of covering wounds with polyurethane membranes produced by electrospinning. These membranes increased the skin regrowth rate and formed a well-organized dermis. The electrospun nano-fibrous membrane could control water loss by evaporation, was permeable to oxygen, and promoted fluid drainage ability, while inhibited external microorganism invasion — all characteristics of healthy skin.

Human dermal fibroblast can be cultured on electrospun nano-fibrous membrane to create in vitro allogeneic dermal substitutes.

Fibroblasts are the cell type best indicated for wound healing proposes. In fact, “planting” fibroblasts into dermal substitutes have been shown to improve wound healing. In this respect, nano-fibrous scaffolds provided enough space for fibroblast ingrowth and induced the formation of a dermal substitute.

In addition, the incorporation of collagen to the nano-fibrous membrane improved attachment and proliferation of fibroblasts.

Other studies have used a scaffold with mesenchymal stem cells (MSCs), and found an increased proliferation rate and differentiation of MSCs when combining with a biomaterial.

In another study, three-dimensional chitosan nanofibers were implanted in mice to cover full-thickness skin wounds and were able to induce a faster regeneration of both the epidermis and dermis compartments when compared to other structures such as sponges.

The development of nanotechnology has also allowed the creation of nanoparticles (NPs) that act as a vehicle and carrier of biological factors that induce skin regeneration. In fact, several promising results have been obtained in studies using NPs involving growth factors, thrombin, nitric oxide, opioids or protease inhibitors. For example, thrombin-conjugated iron oxide NPs improved tensile strength of the wounds, thereby indicating a significant acceleration of the healing process.

Synthetic nanoparticles are able to conjugate peptides, growth factors, nitric oxide or other molecules onto the particle surface and act as delivery vehicles.

Section Summary: Nanofibers can be electrospun to cover a certain area of skin to treat it. It is typically covered with polymers, proteins and/or, other nanoparticles to promote skin growth, especially on scaffolds.

Bone Reconstruction

Trauma, pathological degeneration or congenital deformities make bones one of the most commonly transplanted tissues worldwide. Same person bone grafting and bone allografts are the usual treatment for reconstruction of skeletal defects.

However, open surgery involves a considerable risk of morbidity and implant failure in patient population. As a result of these limitations, the engineering of new bone to replace the damaged bone based on synthetic biomaterials such as metals, polymers, porous ceramics, hydroxyapatite, collagen sponges or hydrogels, among others, have been developed in the past few years.

Despite substantial progress, the construction of structures able to provide the suitable physical and biological properties of the bone still presents challenges. Bone is comprised of hierarchically arranged collagen fibrils, hydroxyapatite and proteoglycans. 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 and natural polymers, such as chitosan and silk fibroin. The combination of synthetic and natural materials has also been studied, in fact, electrospun nano-fibrous scaffolds have been shown to significantly induce bone differentiation of human MSCs (stem cells) and the formation of bone minerals.

Diagram explaining how electrospun nanofibers work.

Recently, bioactive macromolecules like nanohydroxyapatite have been introduced on the surface of polymeric nanofibers, and were proven to regulate and improve specific biological functions like adhesion, growth and differentiation of adipose-derived (fats) stem cells.

Among the materials used for bone-reconstruction, PLLA (polylactic acid) 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. PLLA nanofibers are often functionalized to improve their biological performance with peptides such as RGD (Arg-Gly-Asp) with bone relating molecules such as hydroxyapatite; or with proteins such as collagen and the growth factor bone morphogenetic protein 2 (BMP-2).

Current orthopedic implants fail in an appropriate bone-integration limiting implant lifespan. Recent studies are focused in altering the surface topography of materials at nanoscale level to secure integration with the surrounding tissue and to avoid extrusion and movement.

Nanotopography has been shown to influence the type, quantity and conformation of adsorbed protein, and control cellular adhesion to the surface. 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.

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.

Nano-structured implant surfaces are also known to enhance osteoblast (cell that secretes the matrix for bone formation) reactivity. Using a hydrothermal technique, a simple one-step wet chemical method, non-periodic nanostructures have been developed to surface modify metallic titanium implants.

Among the nano-morphologies tested, the nano-leafy pattern showed the strongest influence on protein grasp, in vitro osteoblast cell proliferation and differentiation. In vivo, these nanostructures have also demonstrated a higher percentage of bone contact without producing any inflammatory response. These results point to the importance of specific nano-morphologies in controlling tissue integration.

In another study, the effect on osteoblast differentiation of TiO2 nanofiber meshes, fabricated using an electrospinning method to create different surface micro-roughness and nanofiber diameters, was evaluated. Osteoblast differentiation and local factor production were regulated by both roughness of surface and the nanotopography, indicating that scaffold structural characteristics alone can be used to drive cell differentiation and create a positive bone forming environment without the use of external factors.

Nanotube structures have also been shown to have great potential for bone regeneration. Their shape is very similar to that of the nanofibers with the only difference that the nanotubes are hollow.

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 nano-crystalline hydroxyapatite, and biocompatible hydrogels which increased osteoblast adhesion.

Close up of a carbon nanotube that can be used as a scaffold.

Carbon nanotubes (CNTs) are other suitable scaffold materials that have proved to support osteoblast proliferation. Indeed, 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.

Section Summary: You can use nanofibers coupled with polymers and other organic materials to enhance bone growth and even apply them on scaffolds along with stem cells to promote/heal the bone even quicker.

Nerve Regeneration

Incomplete recovery from peripheral nerve injuries can produce a diversity of negative outcomes, including numbness, impairment of sensory or motor function, the possibility of developing chronic pain, and devastating permanent disability.

The gold standard treatment is surgery, which requires a nerve graft. Nevertheless, many complications are related to this technique including the sacrifice of the donor nerve function, limited availability of donor tissue, and formation of potentially painful neurofibromas (tumors). Consequently, it is needed to develop new strategies to create nerve artificial prosthesis to solve nerve donor-associated complications.

One of the biggest challenges in peripheral nerve tissue engineering is to create an artificial nerve graft that could mimic the extracellular structure and assist in nerve regeneration. Bio-composite nano-fibrous scaffolds made from synthetic and natural polymeric blends provide suitable substrate for tissue engineering and it can be used as nerve guides eliminating the need for those nerve grafts.

Nano-topography or orientation of the fibers within the scaffolds greatly influences the nerve cell morphology and outgrowth, and the alignment of the fibers ensures better contact guidance of the cells. A bioartificial nerve conduit must meet the overall requirements of a suitable bio-scaffold; it must therefore be biodegradable, biocompatible and non-immunogenic.

Furthermore, nerve conduits should also be engineered to achieve specific characteristics such as to possess an adequate tensile strength without compromising flexibility. It is essential to select the appropriate material in order to reproduce the specific characteristic of the native nerve.

In this respect, numerous materials, synthetic and natural, have been tested for manufacturing nerve conduits. Aliphatic polyesters are biocompatible polymers that can be synthesized into fibers via electrospinning.

Recently, composite materials based on the coupling of conductive organic polymers and carbon nanotubes have shown to possess properties of the individual components and the benefit of a synergistic effect. For instance, multi-wall carbon nanotube (MWCNT)/polymer composites are hybrid materials that combine numerous mechanical, electrical and chemical properties and can be used for the development of nerve guidance channels to promote nerve regeneration.

This biomaterial is a suitable substrate that increases electronic interfacing between neurons and can be employed to create micro-machined electrodes with potential applications in neural regeneration, prosthetic devices and brain implants.

The use of micro-electromechanical systems stimulation, through modulation of ions around the nerve, is a novel nanotechnology strategy for modulating nerve impulse activation. These findings have potentially significant implications for the design of special nano-enhanced materials that could be used to promote nerve regeneration and rehabilitation.

Structure of a nerve cell for reference.

Bridging larger nerve gaps between proximal and distal ends requires tubular constructs with one axis aligned with cues to promote the axonal regrowth. In this respect, electrospun nano-fibrous scaffolds are a good candidate to fill up the gap of the injured nerve. Recently, it has been demonstrated that the alignment of nanofibers has a significant influence on the adhesion and proliferation of Schwann cells.

The axially aligned nanofibers were shown to mimic the fibrin cable architecture and, thereby, this approach may represent an ideal scaffold for extending the growth of axonal processes. Additionally, flexible nerve agent sensors, based on nanotubes with surface substructures such as nano-nodules and nanorods have been explored.

The surface substructures can be grown on a nanofiber surface by controlling critical synthetic conditions during vapor deposition polymerization on the polymer nanotemplate, leading to the formation of multidimensional conducting polymer nanostructures where hydroxyl groups are found to interact with the nerve agents.

Section Summary: Nerve damage is a huge issue which takes a very long time to regenerate and get back to normal. To battle this, the use of nanotubes (either with biomaterials or carbon) can be used to simulate the conductive properties of nerves, and thus help to replace the nerve itself.

Healing Cardiac Tissue

Heart stroke and heart disease are a significant cause of morbidity and mortality worldwide. Heart attack results in reduced cardiac function due to cardiomyocyte (muscle cells that make up the heart) death. As the growth potential of the terminally differentiated cardiomyocytes is low, the heart is unable to repair itself and, after damage, non-functional scar tissue is formed.

Diagram of a heart for reference.

On the other hand, damage or defects in one of the four heart valves can ultimately lead to heart failure. Classical replacement surgery involves the implantation of mechanical valves or biological valves (xeno- or homografts).

Engineering the heart represents a real challenge for a new branch of researchers whose goal is regenerating the damaged cardiac tissue. Certainly, it is not a simple matter of patching the damaged tissue. The elasticity and contractive properties of this “perfect pump” have to be guaranteed in order to avoid complications, such as arrhythmias or dysfunction, which could prevent the correct impulsion of blood to the entire body.

Cell injection directly into the heart has proven to revolutionize the treatment of heart disease. Some clinical trials have been conducted injecting stem cells derived from bone marrow, and some benefits have been proven. Moreover, enhancement in myocardial oxygen consumption and in the contractility properties of the scarred area has been demonstrated.

Considering the drawbacks of cell implantation and the fact that many cardiovascular diseases can lead to heart damage from the necessarily replaced functional structures of the heart — such as valves, or even the whole heart — there is a great need for approaches that create cardiac tissue via bioengineering.

Advances in nanotechnology have allowed researchers to fabricate scaffolds with the aim to mimic the natural cell environment with the physical properties that influence the physiological behavior of the tissue. Thus far, contractile cardiac grafts have been created in vitro and are postulated as a system for replacing dead myocardium and to enhance cardiac function. Furthermore, tissue engineering of heart valves or injection of nanomaterials to improve the function of faulty heart valves, are newly emerging alternatives that improve current modes of therapy in heart surgery.

The use of a degradable, nano-fibrous scaffold made by electrostatic fiber spinning have been suggested to be a feasible method to produce cardiac grafts with clinically relevant dimensions. A variety of biomaterials have been tested, alone or in combinations, to fulfill the requirements of myocardial regeneration.

The priority is to find polymers with specific elastic and ductile mechanical properties that can, for example, mimic the necessary properties of cardiac tissue with the combination of PCL and gelatin, and be properly oriented, yield excellent results and improve adhesion and alignment of cardiomyocytes in the nano-fibrous mesh.

Researchers have developed a biocompatible scaffold that not only has good physical, chemical and mechanical properties, but also present the ability to differentiate cells to cardiomyocytes. In this respect, scientists have created a scaffold combining PEG-PCL-CPCL with an inhibitor of bone morphogenetic protein (BMP) which promotes differentiation of ESCs toward functional cardiomyocytes.

In addition, it has been shown that a scaffold that combines fibrin and PLGA was able to stimulate cardiomyocyte MSC (stem cells) differentiation. Other studies have used a chitosan nanoscaffold coated with fibronectin and proposed a cardiomyocyte-fibroblast co-culture system that resulted in a cardiac tissue-like structure, where cardiomyocytes maintained their morphology and polarity and contracted synchronously.

Recently, cardiac patches of PEG nanoscaffold, embedded in a fibrin hydrogel together with cardiac progenitor cells, were implanted in the ventricle wall of a rat infarction model. The engraftment improves the infarcted area, increasing cell viability and ECM collagen organization.

Another nanotechnological variant consists in the use of NPs, which present important advantages for a targeted therapy. For instance NPs can easily cross the endothelium, and can be administered by a non-invasive procedure, intravenously or by inhalation. Numerous in vivo studies have illustrated the advantages of the use of NPs as complementary therapy in cardiovascular diseases.

The two main nanotechnological strategies for heart valve disease treatment are: (a) the use of tissue engineering to produce fully functional heart valve; or (b) the employment of NPs to alter the physical structure and behavior of faulty valves.

Biological valves are used for valve replacement in surgical therapy for end-stage valvular diseases. But biological prostheses lead to complications such as limited lifespan of the implant due to deterioration and calcification of the valve structure, together with problems related to immunological response.

Nano-engineered heart valves are a promising approach to overcome the limitations of conventional heart valve prostheses. In fact, these valves have been tested in animal models showing excellent tissue remodeling. For instance, fibrin scaffold in combination with arterial-derived cells were inserted in a sheep model and, after three months, the fibrin scaffold was replaced by new tissue containing mature collagen along with functional blood vessels.

One important aspect that has much to do with the efficiency of artificial valves is the procedure employed by culture cells on the scaffolds. In a very recent study, researchers have evaluated which culture conditions are optimal for seeding cells onto polyurethane heart valve scaffolds. They compared static cultivation with dynamic cultivation using a conditioning bioreactor.

Bioreactors are devices in which biological and/or biochemical processes are manipulated through close control of environmental and process-bound factors such as pH, temperature, pressure, and nutrient and waste flow.

After growing endothelial cells and fibroblasts onto the valve scaffolds they evaluated cell confluence, extracellular structure formation and inflammatory response. The study concluded that the use of the bioreactor improved cell attachment to the polyurethane structure and the mechanical properties of the valve scaffold. In these examples, cell growth is supported in vitro and later cell-containing scaffolds are grafted to the animal model.

Natural tissue engineering represents a new approach in which scaffolds are implanted and a signaling component presented in the functionalized scaffold guide cell homing, adhesion and growth. The ultimate goal is to achieve complete cellularization of the graft, the production of a new structure and, finally, tissue formation. Although this type of tissue engineering for heart valve reconstruction is an attractive alternative, more studies are needed to clarify the in vivo feasibility of the approach.

A quick overview on how nanoparticle drug delivery works.

The second main approach based on nanotechnology directed to improve heart valve diseases conditions is the use of NPs. Atherosclerosis processes that involve heart valve degeneration are the target of functionalized NPs that can be used as a vehicle for drug delivery. In addition, NPs can be used to reduce the risk of blood clotting events after heart valve surgery.

Section Summary: Heart disease and other closely related issues are huge problems of death in the world. Often times, the tissue/cells die around the heart resulting in poor performance. Nanoparticles and scaffolds covered with nanomaterials can be used to help stimulate cell growth and heal tissue.

Conclusion

The field of nanotechnology is advancing quickly. This interdisciplinary approach is leading to a rapid expansion and development in the fabrication of biomimetic scaffolds for tissue engineering.

Many studies have been conducted in the search for appropriate materials to create a scaffold that may play an active role in the regeneration process instead of simply being a cell carrier or tissue template. 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. Although some successful devices 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.

Nanotechnologists, cell biologists, and medical doctors have begun to walk the path toward a personalized medicine with the hope of improving the treatment of many diseases. The advanced applications of this approach to regenerative medicine will undoubtedly transform the fundamentals of diagnosis, treatment, and prevention of diseases, becoming an inevitable part of our life.

🔑 Key Takeaways

A quick overview of this lengthy article for those who don’t want to read through it all:

  • Nanotechnology is a coming-of-age tech that can be used to solve some big problems in healthcare
  • Many people suffer from skin wounds, bone destruction, nerve damage, and cardiac tissue death, which all result in long healing times, and sometimes even surgeries that can have many complications
  • Nanomaterials can be used to solve the issues in regenerative medicine to provide a faster, more effect healing method to help patients who suffer from such conditions
  • In most methods, nanofibers are electrospun and then coated with some organic compound
  • Those specialized nanofibers are then put on scaffolds which is then put in the target region in the body to promote cell growth

Overall, this is a fast growing field, with many challenges that still need to be resolved if we want to see this technology in the near future. However, many labs have been able to see positive results and are coming close to making it an option for patients in need.

Thanks for reading, and as always, if you have any questions, comments, or opportunities, feel free to contact me at akashapatel2006@gmail.com or message me through my LinkedIn

See you in the next one!

Innovator | Thinker | Creator. Passionate about medicine and its nanotechnological implications. Working on executing an idea. Website: akashapatel.com