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Bone Tissue Engineering - Why Is Hierarchical Scaffolds Needed?

Osteoporosis, osteoarthritis, trauma-induced injuries, and osteochondral lesions may all be treated using bone tissue engineering (TE), which includes regenerative systems for articular cartilage, meniscal cartilage, ligament, and bone.

In TE, human cells are seeded and attached to a scaffold in vitro. The scaffold creates a favorable microenvironment for the cells to multiply, migrate, and differentiate into a particular tissue type. Therefore, it is clear that the success of TE depends on how this scaffold is designed.

The hierarchical structure of the target tissue must be able to be replicated by scaffold design methods.

Hierarchical in this context refers to the need for the scaffold's properties at length scales ranging from the nanometer to the millimeter, centimeter, and meter levels to match the intended tissue. These properties also determine how well the scaffold satisfies the competing demands of bioactivity and appropriate mechanical and mass transport (permeability and diffusion) needs.

In the case of bone, the macro-scale organization (osteons, osteoids, and Haversian canals) provides the mechanical anisotropy of long bones, while the nano-scale characteristics (collagen type, cross-linking, and hydroxyapatite [HAp] stoichiometry) play a role in cell and mineral binding.

Bone Architecture

Bone and other biological organisms have developed hierarchical three-dimensional structures. It takes a clear understanding of each level to replicate these intricate hierarchical structures in order to replace them.

The two main types of bony tissue—trabecular and cortical—have different anatomical shapes on a macroscopic level. They share a similar composition, but because of their unique hierarchical structuring, they have a very different set of characteristics (such as mechanical characteristics).

Mesoscopically, trabecular bone is composed of plates (trabeculae) that range in thickness from 100 to 300 micrometers and are spaced apart by 300 to 1500 micrometers. These plates are situated next to tiny cavities that are home to red bone marrow. This can be used to create collagen-HAp composite materials that have characteristics similar to trabecular bone, such as collagen fibers that are oriented unidirectionally.

Cortical bone has only 10% porosity and comprises osteons, bundles of mineralized collagen fibers 200m in diameter and 10.0 to 20.0 mm long. The central canal (Haversian canal) contains blood vessels, and small channels (canali-culi) provide microcirculation to the cells. In this system, cells aren't found beyond 200 |im of oxygen and blood. Porosity and channels help with cell migration and vascularization in micro and meso-scale scaffolds.

On a microscopic and molecular level, osteons create bone's extracellular matrix (ECM), a composite of organic and inorganic phases. The organic matrix is mostly collagen type I. Carbonate-substituted HAp mineralizes this matrix. Each constituent has different orientations at nano to micro-scales.

Since cell cytoskeleton and ECM proteins bind directly to cell receptors, cells sense and respond to the matrix's physio-chemical properties by gene expression and protein production. Molecular (collagen type and HAp stoichiometry) and microscopic (microstructure and porosity) scaffold characteristics can affect cell behavior.

Molecular Level

A biomimetic collagen-HAp scaffold's type of collagen, cross-linking, and HAp stoichiometry must mimic bone.


Collagen ligands promote cell attachment, proliferation, and growth factor delivery. Type I collagen's Arg-Gly-Asp (RGD) and Asp-Gly-Glu-Ala (DGEA) peptide sequences promote osteoblast and fibroblast adhesion.

At the molecular level, collagen has three structures: primary, secondary, and tertiary.

Primary collagen structure is (X-Y-Gly), where X is proline and Y is hydroxyproline. These amino acid blocks form a helical polypeptide chain called an α-chain. Their secondary structure is the local configuration of these chains. Tertiary structure indicates large-scale folding and helicity of collagen's chains. Each tropocollagen molecule consists of three-chains forming a triple helix.

Type I tropocollagen, the dominant type in bone, is a triple-helix of two α1(I) and one α2(I) chains (300 nm long and 1.5 nm in diameter), while Type II collagen in cartilage has three α1(II) chains. Monomeric collagen triple helices are telopeptide-deficient (atelocollagen) or intact (tropocollagen).

These triple helices self-assemble into collagen fibrils, which are stabilized by covalent cross-links. The HAp crystals are deposited parallel to the collagen fibers and fill the gaps. Bundles of microfibrils form larger fibrils, which form bigger fibers.


Interactions between hydrophobic and polar groups of collagen molecules assemble the fibrils but don't give the structure much mechanical stability. This is done by intra or inter-molecular covalent cross-linking. Different degrees of cross-linking allow scaffold degradation time to be tailored by improving mechanical properties and enzymatic resistance.

Cross-linking can be chemical (GTA, NHS), physical (DHT, UV), or biological (ENZ). Some of these methods have adverse effects on cell attachment and proliferation on collagen-based biomedical implants (e.g., DHT causes slight collagen denaturation or GTA is cytotoxic).

Therefore, treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) has become a preferred method for collagen crosslinking due to its good biocompatibility, increased mechanical strength, and resistance to enzymatic degradation.


Molecularly, HAp is 40 to 60 nm long, 20 nm wide, and 1.5 to 5 nm thick. They align so the larger crystal dimension is along the collagen fibre axis.

Cain (PO4)6 OH2 is Bone Hap's formula. Due to impurities, including carbonate ions in the lattice, the calcium-phosphorus molar ratio varies. Carbonate ions can replace hydroxide (Type A substitution) or phosphate (Type B substitution), with sodium replacing calcium ions to maintain charge balance. This ionic substitution destabilizes the crystal lattice, making HAp easier to absorb. HAp must be resorbable for bone remodeling. Stoichiometric HA is used as a scaffold, but it has slow degradation.

The particle size of HAp affects osteogenic cells. Small particles aid cellular proliferation.

Microscopic Level

Trabecular bone is porous (50%–90%) and has pores 1 mm in diameter; cortical bone surrounds it. Cortical bone is solid with voids (Haversian canals), resulting in 3 to 12% porosity.

Bone microstructures allow cell migration, nutrient delivery, and vascularization. Pores and their interconnectivity are indispensable for tissue formation. Combining multi-scaled pore size and porosity in one structure improves the overall performance of TE scaffolds. While macroporosity (pore size 450μm) affects osteogenesis, microporosity (10-100 μm) and pore wall roughness are also important.

Mesoscopic Level

Bone formation requires high mass transfer during cultivation, and blood vessels supply it with chemicals. In vivo, cells are 100μm from an oxygen and nutrient-supplying capillary.

The microscopic hierarchy provides cells with interconnected porosity to attach, migrate, proliferate, receive oxygen and nutrients, and dispose of waste. Not all pores are connected. Even if they are fully interconnected with the required dimensions, metabolically active cells cannot migrate deep into the scaffold due to diffusion constraints of oxygen and nutrients; only cells close to the surface can survive, and mineralization at the periphery of the scaffold blocks further diffusion and mass transfer to the interior of the scaffold, leading to growth of only thin cross-sections of tissue (500μm).

For diffusion-related limitations, two general methods can be used. To get cells to go deeper into the scaffold, increase the effective diffusive length. By increasing porosity with hydrogels or specialized carriers (e.g. perfluorocarbons), the diffusion distance can be increased.

Utilizing perfusion to move nutrients and oxygen closer to the cells is another strategy.

Both in vivo perfusion through the cardiovascular system and in vitro perfusion through a perfusion bioreactor are possible. In both situations, using pre-vascularized constructs with microchannels may improve perfusion and vascularization.

Macroscopic Level

It will frequently be necessary to produce the tissue-engineered structures with intricate three-dimensional anatomical shapes. In order to maintain the hierarchy at different levels, the final level of hierarchy needs a processing method for scaffolds that replicates the anatomical shape of the tissue and can be customized for different patients.

TE scaffolds have been created using solvent casting, particulate leaching phase separation, freeze-drying, critical point drying, and SFF. SFF, which uses computerized tomography (CT) or magnetic resonance imaging to acquire voxel data of anatomical structures in order to create a mold for a patient-tailored scaffold, is one of the most promising techniques. The mold is then created using a 3D phase-change printer, and a collagen dispersion with or without HAp is cast and frozen inside of it. It is then critical-point dried after the scaffold has been removed by dissolving the mold.

Fully labeled human leg bone showing the bone tissues
Fully labeled human leg bone showing the bone tissues

People Also Ask

Why Scaffold Is Importance In Tissue Engineering?

Mechanical property: Scaffolds help to stabilize the tissue defect's shape and provide mechanical support. The host tissue's mechanical properties should match those of the biomaterials used as scaffolds or for their post-processing.

What Are Bone Scaffolds Used For?

A bone scaffold is a three-dimensional matrix that encourages osteoinducible cells to attach to and proliferate on its surface.

What Is The Purpose Of A Scaffold In Regenerative Medicine?

The creation of a 3D environment to encourage tissue formation relies heavily on biodegradable scaffolds. A significant amount of potential for tissue regeneration is thought to exist when scaffolding materials are used in conjunction with stem cell technologies.

Do Bones Provide Scaffolding?

Collagen (and gelatin), which make up the majority of bone, are the perfect materials for 3D scaffold design. It promotes cell proliferation and differentiation as an extracellular matrix and is inherently biocompatible and biodegradable.


In order to create templates that are best suited for bone tissue engineering, a scaffold's hierarchical design may be used to mimic the structure of extremely complex tissues like bone. It has been discussed how structuring a bone scaffold under control during manufacturing at each level of hierarchy can aid in such design. The bioactivity, degradation, and mechanical properties are improved at the molecular level by choosing the proper collagen (Type-I monomeric collagen)-HAp scaffold with the appropriate stoichiometry and cross-linking. Pore size and porosity, which at the microscopic level affect cell response and mechanical properties, can be tailored by using an appropriate fabrication method and changing the parameters. Incorporating channels improves cell in-growth and vascularization at the mesoscopic level, and at the macroscopic level, creating anatomically shaped scaffolds using techniques like SFF can result in patient-tailored scaffolds.

About The Authors

Suleman Shah

Suleman Shah - Suleman Shah is a researcher and freelance writer. As a researcher, he has worked with MNS University of Agriculture, Multan (Pakistan) and Texas A & M University (USA). He regularly writes science articles and blogs for science news website immersse.com and open access publishers OA Publishing London and Scientific Times. He loves to keep himself updated on scientific developments and convert these developments into everyday language to update the readers about the developments in the scientific era. His primary research focus is Plant sciences, and he contributed to this field by publishing his research in scientific journals and presenting his work at many Conferences. Shah graduated from the University of Agriculture Faisalabad (Pakistan) and started his professional carrier with Jaffer Agro Services and later with the Agriculture Department of the Government of Pakistan. His research interest compelled and attracted him to proceed with his carrier in Plant sciences research. So, he started his Ph.D. in Soil Science at MNS University of Agriculture Multan (Pakistan). Later, he started working as a visiting scholar with Texas A&M University (USA). Shah’s experience with big Open Excess publishers like Springers, Frontiers, MDPI, etc., testified to his belief in Open Access as a barrier-removing mechanism between researchers and the readers of their research. Shah believes that Open Access is revolutionizing the publication process and benefitting research in all fields.

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