“ An interdisciplinary field of research that applies the principles of engineering and the life sciences towards the development of biological substitutes that restore, maintain, or improve tissue function. ” –Langer and Vacanti*
Tissue Scaffolding Tissue scaffolds are structures that are made of either artificial or natural substances that act as a guide to help cells grown and expand. It is the bare bone structure that cells will adhere and grow on to to help form the tissue being reginerated. The scaffold can either be inert or can actively help the cells grow through the release of chemical signals. Scaffolds can be constructed from various materials some of which include:
- Calcium Phosphate ceramics
- Silk proteins
A tissue scaffold needs to be porous. Pores allow the cells to interconnect and adhere to one another. Ideally, the scaffold should also release chemicals that help to promote cell migration, cell adhesion and differentiation into specialized cells.
Fugure 2. Process of Producing a Tissue Engineering Scaffold
Scaffolding for Tendons Two main types of tendon scaffolding are predominantly on the market, the two types are biological and synthetic scaffolding. Biological scaffolds are made from organic materials that hold the collagen while it grows into place. Have an organic scaffolding for the tendon will reduce the risk of infection. The noncollagen component of the scaffold is reduced through a cascade of steps which include cleaning, removal of fat deposits, crosslinking and sterilization. The remaining parts of the scaffold are the parts that are comprised of the naturally occuring collagen fibers that can be found within the body. Synthetic scaffolds are of particular interest in that certain materials may cause an inflammatory response or may even be rejected by the host. One scaffolding material that comes to mind is the synthetic extracellular matrix or ECM grafts for short. ECM’s are adequate scaffolds for cellular and fibrotic growth and also it reduces the risk of provoking an inflammatory response.
Figure 3. Example of a Bone-Tendon Junction using a Scaffold
Allogeneic tendon-bone scaffolds can be used as engraftments in the upper and lower extremities. The use of these scaffolds would allow reconstruction with bone-to-bone and tendon-to-tendon healing, creating stronger junctions at an earlier time point.
This technology is based on decellularizing human tendon-bone interfaces by a process that greatly reduces the cellular content and nucleic acid content of the tendon and bone while preserving the mechanical strength and biological properties of the strong anchoring junction. The lab has demonstrated that this decellularized composite tissue is a viable scaffold for the growth and proliferation of human cells, including dermal fibroblasts and mesenchymal stem cells. Next steps are to develop techniques to enhance the growth, proliferation, and migration of allogeneic cells seeded onto the decellularized scaffold with the goal of promoting more rapid tendon engraftment and restoration of pre-injury strength and mechanics.
- Tissue reconstruction of upper and lower limbs:
- Upper limb – Rotator cuff Zone I, Flexor tendon repairs at TBI, Elbow collateral ligament, Biceps attachment, scapholunate ligament, collateral ligaments
- Lower limb – ACL, Achilles tendon, Knee collateral ligament
- Faster healing time
- Maintains strength – New methods described in this invention have led to decellularization of the tendon-bone interface
- Repairs tendon– bone interface (TBI) which is a uniquely strong junction in human tissues that is extremely difficult to reconstruct
- Improves upon existing repair/reconstruction techniques such as suture anchor / Drill holes in bone
- Uses ready-made, off-the-shelf tissue engineered TBI grafts
- Possibility of latent disease/illness with the use of foreign tissues/cells in scaffolding
- Ethical concerns with in vitro tissue/cell growth as well as stem cell research
Typical Injuries Requiring Tissue Junction
Tendon or tissue junction injuries can occur in various ways. An example of this would be a tommy john injury or a torn MCL or ACL where a junction or replacement ligament might be needed in order to fully heal the injury. Growing the tendons on a scaffold would allow for a more precise fit for the new tendon to the body.This interface consists of a band of fibroblast-laden, interdigitating band of tissue that connects the dense collagen fibers of the tendon to the more elastic muscle fibers while displaying a gradient of structural properties. The muscle-tendon junction is another critical research area for integrative tendon repair. As the tendon joins the muscle to bone, which connects muscle to tendon, acts as a bridge to distribute mechanical loads. These engineered scaffolds can also be used for tendon-to-bone junctions such as rotator cuff tear.
[Surgery and implantation of a scaffold to repair a torn ACL, anterior cruciate ligament. ACL surgery is usually done by making small incisions in the knee and inserting instruments for surgery through these incisions (arthroscopic surgery). In some cases, it is done by cutting a large incision in the knee (open surgery).]
Basics to Engineering a Scaffold for Tendon-to-Bone Junctions
Critical Parameters That Define Scaffolds Performance
- High porosity and pore volume to allow for good cell to cell interaction(a minimum pore size between 100 and 150 μm)
- For continuous in-growth of bone tissue, interconnected porosity is important. Open and interconnected pores allow nutrients and molecules to transport to inner parts of a scaffold to facilitate cell in-growth, vascularization, as well as waste material removal
- Since higher porosity increases surface area per unit volume, the biodegradation kinetics of scaffolds can be influenced by varying pore parameters
- Biodegradation through a cell-mediated process or chemical dissolution are both important to ascertain stabilized repair and scaffold replacement with new bone without any remnant
- Initial mechanical properties and strength degradation rate should match that of the host tissue for optimum healing
- Encourage good cell organization and cell to cell interaction
- Be approved by body to avoid inflammation
- Geometry of structure
- Good cell adherence
- Degradation rate
Based on observations, the ideal scaffold for tendon-to-bone interface tissue engineering must exhibit a gradient of structural and mechanical properties mimicking those of the multi-tissue insertion. Compared to a homogenous structure, a stratified scaffold with pre-designed, tissue-specific matrix inhomogeneity can better sustain and transmit the distribution of complex loads inherent at the direct insertion site. A key criterion in stratified scaffold design is that the phases must be interconnected and pre-integrated with each other, thereby supporting the formation of distinct yet continuous multi-tissue regions. In other words, the scaffold would exhibit a gradient of physical properties in order to allow for the recapitulation of interface-like heterogeneity throughout the scaffold. It should also support growth and differentiation, as well as the interactions between heterotypic and homotypic cell populations to promote the formation and maintenance of multi-tissue interface. In addition, the scaffold phases should be biodegradable so it is gradually replaced by living tissue, and the degradation process must be balanced with respect to mechanical properties in order to permit physiological loading and neo-interface function. Finally, the interface scaffold must be compatible with existing tendon reconstruction grafts or pre-incorporated into tissue engineered graft design in order to achieve integrative and functional soft tissue repair.
Examples of Materials used for Construction of Scaffold
- Natural Biomaterials– the different components that make up the extracellular matrix provide a starting point for developing scaffolds based on natural biomaterials. These proteins and polysaccharides perform many roles in vivo and thus make such materials attractive for tissue engineering applications. Additionally, their natural origin often means that these materials contain sites for cellular adhesion and tend to be biocompatible. Some of the disadvantages of these materials include potential lot to lot variability of the material, depending on the source, as well as needing to ensure the purity of the protein or polysaccharide before implantation to avoid activating an immune response. These scaffolds often have a limited range of mechanical properties and may need to be optimized for stem cell culture.
Ex: Silk, Fibrin, Collagen
- Synthetic Biomaterials– synthetic biomaterials provide an alternative to natural materials to serve as scaffolds for the culture of stem cells. These materials offer many advantages including reproducibility due to their defined chemical composition and the ability to control the mechanical properties, degradation rate, and shape independently. The mechanical properties of a scaffold can influence the resulting stem cell differentiation. The ability to shape a material allows for production of scaffolds that conform to specifications of the injury or transplantation site. The ability to tailor scaffolds with specific degradation rate is one advantage of synthetic scaffolds over natural biomaterials and these properties can also affect the release rate of drugs incorporated into such scaffolds. However, many of the synthetic materials lack sites for cell adhesion and may have to be chemically modified to contain such cues to allow for stem cell adhesion and culture. Other considerations include the biocompatibility of the material and its suitability for transplantation in vivo, as well as whether or not the material and its byproducts can trigger an immune response.
Examples of Synthetic Biomaterials:
- Poly (lactic-co-glycolic acid) – Poly (lactic-co-glycolic acid) (PLGA) is a copolymer that consists of monomers of glycolic acid and lactic acid connected by ester bonds. It is an FDA approved polymer that is attractive for tissue engineering applications due to its biocompatibility and the ability to modulate the degradation rate. In the presence of cells, PLGA scaffolds degrade in the monomers, which are natural metabolites but can have negative effects due to their acidic nature. For these reasons, PLGA scaffolds have been used for engineering a wide range of tissues.
Figure 4. Designing PLGA scaffolds that mimicks the architecture of the spinal cord.
Table 1. Scaffold-based Approaches for Tendon-to-Bone Interface Junctions(in vtiro and in vivo evaluations) 
|Study||Scaffold Composition||Scaffold Design||Cell type/Evaluation||Observations|
|Erisken et al. 200822||Unaligned, extruded/electrospun PCL nanofibers (200–2000 nm)||Gradient of scaffold mineral content||Mouse preosteoblast cells (MC3T3) in vitro||Formation of a gradient of calcified matrix|
|Li et al. 200951||Gelatin-coated PCL nanofibers and plasma treated PLGA||Graded coating of calcium phosphate||Mouse preosteoblast cells (MC3T3) in vitro||Cells preferentially adhere and proliferate on areas with high CP content|
|Xie et al. 201051||Aligned and unaligned PLGA nanofibers||Aligned-to-random fiber orientation||Rat tendon fibroblasts in vitro||Cells align/organize on aligned fibers; cells remain unorganized on random fibers|
|Moffat et al. 201162||Aligned PLGA deposited over PLGA-HA nanofibers Phase A (615±152nm) Phase B (340±77nm)||Biphasic with contiguous layer of PLGA and PLGA-HA||Bovine chondrocytes in vitro Rat BMSC in vivo Rat rotator cuff model||Contiguous non-calcified and calcified fibrocartilage-like matrix were formed in vitro and in vivo|
Table 2. Techniques for bone/tissue engineering scaffold fabrication
|Technique||Process details||Processed materials for bone tissue engineering||Advantages (+) and disadvantages (−)|
|3D Plotting/direct ink writing||→ Strands of paste/viscous material (in solution form) extrusion based on the predesigned structure
→ Layer by layer deposition of strands at constant rate, under specific pressure
→ Disruption of strands according to the tear of speed
→ Hydroxyapatite (HA)
→ Bioactive glasses
→ Mesoporous bioactive glass/alginate composite
→ Polylactic acid (PLA)/polyethylene glycol (PEG)
→ PLA/(PEG)/G5 glass
→ Poly(hydroxymethylglycolide-co-ɛ-caprolactone) (PHMGCL)
→ Bioactive 6P53B glass
→ Mild condition of process allows drug and biomolecules (proteins and living cells) plotting
→ Heating/post-processing needed for some materials restricts the biomolecule incorporation
|Laser-assisted bioprinting (LAB)||→ Coating the desired material on transparent quartz disk (ribbon)
→ Deposition control by laser pulse energy
→ Resolution control by distance between ribbon/substrate, spot size and stage movement
→ HA/MG63 osteoblast-like cell
→ Nano HA
→ Human osteoprogenitor cell
→ Human umbilical vein endothelial cell
→ Ambient condition
→ Applicable for organic, inorganic materials and cells
→ Quantitatively controlled
→ 3D stage movement
→ Homogeneous ribbons needed
|SLS (Selective Laser Sintering)||→ Preparing the powder bed
→ Layer by layer addition of powder
→ Sintering each layer according to the CAD file, using laser source
→ Nano HA
→ Calcium phosphate (CaP)/poly(hydroxybutyrate–co-hydroxyvalerate) (PHBV)
→ Carbonated hydroxyapatite (CHAp)/poly(L-lactic acid) (PLLA)
→ β-Tricalcium phosphate (β-TCP)
→ No need for support
→ No post processing is needed
→ Feature resolution depends on laser beam diameter
|SLA (Stereolithographic)||→ Immersion of platform in a photopolymer liquid
→ Exposure to focused light according to desired design
→ Polymer solidifying at focal point, non-exposed polymer remains liquid,
→ Layer by layer fabrication by platform moving downward
|→ Poly(propylene fumarate) (PPF)/diethyl fumarate (DEF)
→ Complex internal features can be obtained
→ Growth factors, proteins and cell patterning is possible
→ Only applicable for photopolymers
|FDM (Fused deposition modeling)||→ Strands of heated polymer/ceramics extrusion through nozzle||→ Tricalcium phosphate (TCP)
→ TCP/polypropylene (PP)
→ Alumina (Al2O3)
No need for platform/support
→ Material restriction due to need for molten phase
|Robotic assisted deposition/robocasting||→ Direct writing of liquid using a nozzle
→ Consolidation through liquid-to-gel transition
→ 6P53B glass/PCL
→ Independent 3D nozzle movement
→ Precise control on thickness
→ No need for platform/support
→ Material restriction
Example of Company Product on the market
Company: Reinnervate Product: Alvetex
Comparison of Alvetex vs. 2D Scaffold Products
3D vs. 2D Scaffolds: Alvetex Scaffold Product Animation Video
- Alvetex is a highly porous polystyrene scaffold designed to enable accurate reproduction of in vivo cell function and responsiveness within an in vitro model system.
- By accurately re-modelling the dimensions and environment of living tissues Alvetex enables cells to maintain natural morphology and tissue-like organization. Cells grown in Alvetex possess a natural tissue-like structure that enables cells to function in a more physiologically relevant manner.
- Alvetex maintains natural cell shape and morophology. In conventional 2D cell culture, cells come into contact with the flat surface of the culture vessel (e.g. Petri dish, flask or multi-well plate). In this unnatural environment cells become flattened against the substrate. In this abnormally thin structure there has been significant re-modelling of the internal cellular components. The entire cytoskeleton is re-modelled and organelles such as the nucleus are flattened.
- Alvetex not only preserves the native 3D shape of individual cells, it also acts to bring cells together in a more natural manner. This results in the formation of tissue-like structures and cell-to-cell interactions that are more representative of normal tissue function.
- Alvetex is the ideal format to enhance the physiological relevance of your in vitro model and create tissue-like structures in vitro with multiple cell types. Cells of different types can be brought together in 3D co-culture models, either as mixed populations or as discrete layers of different cell types.
Figure 6. Co-Culture Models
Figure 7. Comparison of the Structure of Fibroblasts in conventional 2D cell culture and 3D cell culture using Alvetex Scaffold
– Confocal imaging of cells (fibroblasts) grown in 2D (left image) and in Alvetex Scaffold (right image) revealing the dramatic differences between 2D and 3D growth. In the 2D model when looking from above the outline of the cell is normal but from the side one can see how the cell has become very flattened. The same applies to the nucleus where looking from the side one can see how flattened the nucleus become. In contrast the profile of the cell cytoskeleton and call nucleus either form top or side when viewed in the Alvetex Scaffold is able to maintains its natural spherical shape. There is extensive evidence to show that cell shape modifies gene expression, protein translation and in turn function and this is widely reported in the literature including the two articles detailed.
Figure 8. In Vitro results of Cell Reproduction and Growth of Alvetex Scaffold
Figure 9. In Vivo Results of Alvetex Scaffold
Video on Inter Cellular Tissue Junctions- The Tissue Level of Organization
[Video describes the the four main inter cellular tissue junctions and the organization of each and how they each work]
Issues With Tissue Engineered Scaffolds
Increasing the porosity will decrease the strength of the scaffolds. Low strength along with brittleness makes these scaffolds difficult to even handle during processing. Resorbable polymer infiltration to enhance strength and toughness in these scaffolds is one way to minimize this problem; the use of resorbable glassy materials can also help. Finally, printing live cells or adding growth factors/drugs is another fascinating area of growth. However, most of the challenges here are limited to survivability of the cells, viability of the growth factors and drugs after printing. Although current techniques let us build structures with similar composition to that of tissue, we are still a long way from completely printing functioning tissue. 
Research for the future of tissue junction scaffolding
1. Co-electrospun muscle-tendon dual scaffolding:
- Single tissue scaffolds have been around for a little while, but the need for composite scaffolds is high. In order to have a proper muscle-tendon junction scaffold, there needs to be a seamless interface that can handle the force transfer from the muscle to the tendons. In order to do this the scaffold has to be able to take on the properties of both tissues. Using a co-electrospun dual scaffold allows for similar characteristics to an actual muscle-tendon junction. Ladd et. al have decided to use Poly(ε-caprolactone)/collagen and poly(l-lactide)/collagen to create a dual scaffolding. After testing both a native muscle-tendon junction as well as the scaffolding, they were able to deduce that the scaffolding showed similar properties and characteristics to a normal muscle-tendon junction.
2. Tendon tissue engineering using scaffold enhancing strategies:
- Liu et. al have noticed that tendon disease are common and because of this they have decided to engineer substitutes to put in the place of the real thing. There have been three categories of materials used for scaffolds including polyesters, polysaccharides, and collagen derivatives. Some strategies that have developed to enhance neo-tendogenesis include cellular hybridization, interfacing improvement, and physical stimulation. There are methods that can repair damaged tendons, including using sutures and soft tissue anchors, but these repaired tendons are still not as strong or useful as uninjured tendons. There are some alternative therapies for tendon repair, including biological grafts, permanent artificial prostheses, and tissue engineering. Tendon tissue engineering is probably the best approach to fixing the tendons because it does not involve using foreign substitutes. Instead it uses a scaffold that temporarily sits there to help support initial tissue growth. The scaffold can also promote matrix production and enhance tendogenesis. These can be achieved by cell proliferation, cellular hybridization, surface modification, growth factor cure, mechanical stimulation and contact guidance.
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