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3D Printing: Successful Scaffolds in Bone Regeneration

In ‘Comprehensive Review on Full Bone Regeneration through 3D Printing Approaches,’ the authors review new developments and solutions in tissue engineering for the formation of cells, as well as proposing an optimized temporary support geometry for treatment.

Fused Filament Fabrication (FFF) process.

Bone regeneration continues to challenge researchers in their work as well as medical professionals attempting to improve patient treatment:

“Many research groups have been working on bone regeneration for over 10 years, but this has not led to effective therapy in a clinical setting. If it was successful, it would enhance the quality of life for millions of people and significantly reduce the absence to work due to fractures which are considered the second higher cause of working day lost,” state the authors.

“When there are fractures with a bone defect exceeding a critical size, the bone is not able to self-regenerate and, therefore, requires the use of a temporary implant (natural and/or synthetic) to serve as support and cells to help bone regeneration. In this way, tissue engineering (TE) has emerged.”

While scaffolds are used in tissue engineering for transporting nutrients and secretion of waste, the cells must be able to imitate true tissue biology, morphology, and functionality.

Exploring the usefulness of temporary implants, the authors state that in tissue engineering for patients, it is first critical to examine native bone tissue and mechanical properties.

Human long bone properties.

3D printed implants must be able to sustain cell viability in a secure environment, and scaffolds must possess suitable elasticity for matching regular bone. High porosity is desired in most tissue engineering, along with the use of materials that are not only biocompatible but also biologically active. During trials, animal models of fractures are often used in vivo before procedures are attempted on humans.

“Animal studies are needed to understand bone regeneration. Variables such as the amount of bone formation and its kinetics, mechanical properties and safety obtained by the scaffold, including the presence of toxic degradation in different organs and in terms of inflammatory response need to be understood in detail,” explained the researchers.

“However, bone fractures performed in animals do not represent the complexity of healing human fractures. The potential of each different type of cells both in vitro and in vivo plays here a key role.”

Even more interesting though, the authors point out that growth factors are unnecessary, with cells showing the potential to secrete optimal extracellular matrix (ECM) components.

“In vitro studies are advantageous because they offer a controlled environment to experimental test molecular and cellular hypotheses,” stated the researchers. “However, cells cultured in vitro are not replicates of their in vivo counterparts.”

While tissue engineering can be a delicate process overall in terms of working to keep cells alive, bone generation is particularly challenging—and scaffolds must be relied on to maintain the same role as tissue. Biomaterials must be able to mimic the natural environment, along with possessing identical mechanical properties of the initial bone. Appropriate levels of degradation are critical for bone regeneration, and are also dependent on corrosion resistance and materials.

Characteristics of the different materials used to produce a scaffold.

Suitable materials include poly(ε-caprolactone) (PCL) or polylactic acid (PLA), both approved by the FDA and offering stability, biocompatibility, and biodegradability. Scaffolds must be osteoinductive for sustaining cells as well as being osteoconductive, providing growth. They must also serve to:

  • Fill bone defect
  • Ensure pore connectivity
  • Encourage bone formation
  • Promote bone growth

Natural organization of long bones.

Designed in SolidWorks, the structures exhibited ‘superior advantages’ over what could be produced conventionally.

“Considering all types of materials available, associated with the desired bone regeneration and the use of synthetic polymers, as PCL or PLA, combined with collagen type I for the trabecular region and Hap for cortical region, seems to be the best strategy to follow,” concluded the researchers.

“Among the most commonly used bioreactors for bone regeneration, perfusion bioreactors appear as the most suitable, because it improves osteogenic proliferation and differentiation due to improved mass transfer and adequate shear stress. When making a design proposal for bone regeneration, it is necessary to study the mechanical effects, such as stress and tension, and link them.”

Cylindrical scaffold

DNA chain-inspired cylindrical scaffold.

Tissue engineering continues to be an enormous area of study, from seeding human dermal fibroblasts, promoting hydrogel microenvironments, to bioprinting structures for soft tissue engineering applications.

Scaffold requirements in terms of response (left) and what should be taken into account (right) (adapted from [106]).

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[Source / Images: ‘Comprehensive Review on Full Bone Regeneration through 3D Printing Approaches’]

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Bone Regeneration: Successful Bioprinting with Poly-Lactic-Co-Glycolic Acid/β-Tricalcium Phosphate Scaffolds

In ‘Poly(Dopamine) Coating on 3D-Printed Poly-Lactic-Co-Glycolic Acid/β-Tricalcium Phosphate Scaffolds for Bone Tissue Engineering,’ researchers from the School of Stomatology at Jilin University in Changchun, China are seeking improved methods for treating bone defects caused by health issues like osteoporosis, malignant tumors, and physical trauma. While currently there are numerous limitations for medical treatment, bioprinting and bone tissue engineering show great potential.

For this study, the researchers 3D printed polydopamine-coated poly-(lactic-co-glycolic acid)/β-tricalcium phosphate composite scaffolds, followed by characterization and biocompatibility tests.

The sample scaffolds created by the research team exhibited a regular shape, and pore size of ~500 μm, with a honeycomb structure—a form not uncommon in bioprinting.

Overall appearance and structure of different scaffolds. (A) 3D-printed PLGA/β-TCP composite scaffolds with different PDA coatings; (B) reconstructed images from the micro-CT of the 3D-printed composite scaffolds with different coatings.

“The printed filaments are exhibited in a network structure, with a strong architectural integrity. The surface morphology of the fiber-aligned scaffolds, with or without PDA surface modification, can be characterized by SEM,” stated the researchers. “The results show that PDA0, PDA1, and PDA2 scaffolds have pores distributed over the internal walls, with a highly roughened structural coating layer and uneven micro-surface in comparison with non-coated scaffolds.”

Surface morphology of different groups of scaffolds, observed under SEM. The images of different scaffolds show the surface features of the PDA0 group, PDA1 group, and PDA2 group at different magnifications. PDA is formed into a film-like coating on the surface of the scaffolds at a high magnification (red arrows).

PDA0, PDA1, and PDA2 were evaluated for surface wettability using water contact angle measurement, with PDA0 exhibiting the highest contact angle at 88.3°, 68.5°, and 55.1°, respectively (p < 0.05). Porosities were found to be 60.31%, 61.1%, and 59.67%, respectively. While porosity can be maintained during bone tissue engineering, density varies

Characterization of the scaffolds from different experimental groups. (A) shows the water contact angle of the surface of the scaffolds of different groups; (B) shows the density and porosity of the scaffolds in different groups, where any differences in statistics are not indicated, and the scaffolds of different groups have a porosity of approximately 60% and density of approximately 0.28 g/cm3; (C) represents the average pore sizes of the scaffolds in each group, which are all approximately 500 μm, without differences; (D) indicates the mechanical properties, including the strength and modulus, and the affiliated figure denotes the trend of the scaffolds in each by a strain–stress curve. The results do not display any differences between the different groups, which are 0.65 MPa in strength and 26.5 MPa in modulus.

For cell attachment tests, the research team used mouse pre-osteogenic cells, demonstrating cell seeding efficiencies of the PDA0, PDA1, and PDA2 scaffolds at 56%, 76%, and 82%, respectively. They also noted that the PDA2 group ranked highest in adhesion (p < 0.05). As the PDA coating increased, cell membranes spread more expansively.

The cell-adhesion properties of the scaffold. (A) shows the adhesion efficiency, when cells were seeded into the scaffold; (B–D) are the pictures under a confocal laser microscope, stained by DAPI, which represent the PDA0, PDA1, and PDA2 groups, respectively.

Ultimately, the PDA-coated PLGA/β-TCP composite scaffolds were 3D printed successfully, according to the research team. Along with successful fabrication efforts, the researchers found that the sample scaffolds were ‘suitable’ as vehicles for bone tissue engineering. Using a one-way ANOVA analysis, the team evaluated the variations between the experimental groups. Results were ‘statistically significant’ at a p value <0.05.

“Furthermore, the in vitro cell biocompatibility test results show that the PDA coatings can improve cell adhesion and osteogenic differentiation. To further study the osteogenic effects of the PDA-coated scaffolds, we established a mouse skull defect model and implanted the scaffolds in the defects for a period,” concluded the authors.

“The bones with scaffolds were extracted to be analyzed for new bone formation, and the results show that the PDA-coated composite scaffolds perform a satisfactory repair effect. In conclusion, our results demonstrate that the simplified bio-inspired surface modification of PLGA/β-TCP scaffolds by PDA is a very promising method for effectively repairing bone defects.”

Because of the difficulties and challenges in bone tissue engineering, there is continued research into the subject, from studies regarding nanofiber tubular scaffolds to structures made with tricalcium phosphate, porous metallic biomaterials, and more.

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[Source / Image: ‘Poly(Dopamine) Coating on 3D-Printed Poly-Lactic-Co-Glycolic Acid/β-Tricalcium Phosphate Scaffolds for Bone Tissue Engineering’]

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Bioprinting for Bone Regeneration with Nanofiber Coated Tubular Scaffolds

Researchers from both Mexico and Costa Rica have joined efforts to further research into bone regeneration via bioprinting, allowing doctors and surgeons to create patient-specific scaffolds for improved treatment. 3D printing and tissue engineering show great promise for scientists because of the opportunity to build complex geometries, with precision. All the classic benefits of 3D printing are enjoyed during these experiments too, like affordability, speed in production, and best of all—the ability to create on-demand in the lab, manufacturing and making changes to structures in a completely self-sustained fashion.

The team of researchers detail their findings further in ‘Biocompatibility of Developing 3D Printed Tubular Scaffold Coated with Nanofibers for Bone Applications’ explaining how bone scaffolds can be improved further with an added composite layer that creates a layer more conducive to cell attachment and uniform seeding. To create these scaffolds, the team used a unique air jet spinning (AJS) technique, featuring a specialized spinning system nozzle and a surface for collecting polymer fibers and compressed gas—and they also 3D printed tubular scaffolds with PLA, featuring ‘submicrometric fiber surface coating in the biological response of human fetal osteoblast cells (hFOB).’

This new method uses both the inner core of the PLA 3D printing material and the outer layer of its nanofibers, with the researchers using Cura software for internal geometries and a MakerMex 3D printer to manufacture the tubular structures. The dual technique allowed the team to create a fiber layer dispersion resulting in a surface with ‘homogeneous thickness distribution’ and nanofibers adapting well to merging with the 3D printed scaffold. Adhesion was noted as ‘very strong,’ with the composites showing an increase in thermal stability, and the coating imbuing the tubular scaffold with properties critical to tissue engineering for bone regeneration.

SEM micrographs showing the morphology of the 3D-printed tubular scaffold

“The 3D surface of the printed tubular scaffold exhibited distinctive morphologies and structures analyzed by SEM, and the surface roughness of the tubular scaffolds increased with the incorporation of the coating functionalization by the fiber membrane,” concluded the authors.

“Moreover, scaffolds coated with submicrometric fibers allow hFOB cells to adhere and proliferate better than uncoated 3D tubular scaffolds showing that the fibers work as a platform to improve cell biocompatibility (being not toxic to cells) and provide support to colonization and cell growth by the osteoblast cells. Moreover, the 3D tubular scaffold coated with fibers needs more studies as a biomineralization process for it to have a potential future use in bone tissue engineering or for it to have an application in the vascularization process.”

Optical profiler data showing the topography of a 3D-printed tubular scaffold. (a) Images show the uncoated smooth surface and
(b) the coated surface where roughness is strongly enhanced by the presence of nanofibers.

The field of bone regeneration is fraught with challenge, but doctors and surgeons press on to make improvements in both surgical techniques and devices such as implants so that they can improve the quality of life for patients who may be debilitated or in great pain. Researchers have engaged in many different studies over the years regarding 3D printing, producing devices such as implants made just for patients in China, bone scaffolds created at low temperatures, and other different types of bone scaffolding platforms. What do you think of this news? Let us know your thoughts! Join the discussion of this and other 3D printing topics at 3DPrintBoard.com.

SEM micrographs of the 3D-printed tubular scaffold coated with 7% PLA nanofibers.

SEM micrographs of the 3D tubular scaffold surface seeded with hFOB cells showing some cells with an oval to spindle-shaped morphology typical of osteoblasts cells.

[Source / Images: ‘Biocompatibility of Developing 3D Printed Tubular Scaffold Coated with Nanofibers for Bone Applications’]

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University of Amsterdam: Researchers 3D Printing Grafts for Alveolar Ridge Augmentation

At the University of Amsterdam, researchers may be seriously impacting reconstructive dentistry with a new process for strengthening the alveolar ridge after tooth loss or other more significant health issues. In ‘Marginal and internal fit of 3D printed resin graft substitutes mimicking alveolar ridge augmentation: An in vitro pilot study,’ authors C. C. Stoop, K. Chatzivasileiou, W. E. R. Berkhout, and D. Wismeijer explain a new design they have engineered for bone regeneration with 3D printed grafts.

As is so often the case with 3D printing, the key is in customization—allowing for patient-specific treatment with a graft meant to apply to both horizontal and vertical augmentation of the atrophic alveolar ridge. With CT scans converted into data for creating completely customized grafts, treatment time is expected to be reduced and there is a greater chance for regeneration. This innovation accentuates the growing reliance of implants and prosthetics in resolving issues with appearance and chewing after tooth loss. Historically, dental implants can be challenging without the ability to customize extensively—and even with more patient-specific treatment, there are many obstacles that can come into play regarding successful implantation and regeneration.

Representative lateral views of virtual 3D models with small-defects.

Lateral views of virtual 3D models with large-defects.

(A) Frontal view of a CAD mandibular model. (B) Lateroinferior view of a CAD large mandibular graft. (C) Frontal view of the graft fitted on the recipient site of the mandible. (D) Frontal CBCT view of the fitted graft.

The amount of bone volume left plays a large role in success, along with the types of defects involved—whether they are due to gum disease, cysts or tumors, teeth being pulled, or trauma to the mouth or jaw. There is also a range of different categories for defects, with the worst scenario being both horizontal and vertical defects occurring simultaneously.

“In situations of major changes in both vertical and horizontal bone dimensions, the placement of dental implants without an augmentation procedure could be very complicated or even impossible,” state the researchers.

Guided bone regeneration is usually a long process, and surgeons rely on a mixture of both biological and mechanical properties. Autogenous bone blocks are often used in surgeries, with grafts secured on the ridge or in between areas of pedicled and internal cancellous bone. The researchers state that success often relies on integration of the blocks as well as how well graft edges are smoothed, their stability, and their shape:

“The ideal biomaterial should be customized to fit easily on or into the corresponding bone defect and to allow proper fixation,” state the researchers. “The optimal shape of the graft may come with multiple advantages in terms of: (A) Faster surgical procedures, (B) Better healing of the grafted site, (C) Reduced risk of peri- and post-operative complications, (D) Higher success rates of the bone augmentation procedure and (E) Higher patient satisfaction.”

Shaping of bone grafts can be complex and requires an experienced hand. Without the proper expertise, healing may be unsuccessful.

“For this reason, there is a clinical need for customized biomaterials shaped to fit the patient’s bone defects,” said the authors.

With the options available through CAD design, bone defects can be scrutinized more closely, and customized, accurate grafts can be created before surgery. The entire process is more streamlined, surgical procedures are faster, and the patient has a better experience all around.

Six patients from the clinic of Oral Implantology and Prosthetic Dentistry at the Academic Centre for Dentistry Amsterdam (ACTA) were involved in the study, with the researchers evaluating cone beam computed tomography (CBCT) datasets from each, to be treated with implants and augmentation with bone blocks. Patients all presented as:

  • Partially dentate
  • Combined vertical and horizontal defect of the maxilla (three patients)
  • Combined vertical and horizontal defect of the mandible (three patients)

3D reconstruction was completed for each patient’s jaw, with data imported into Meshmixer for design:

“The atrophic bone areas were defined separately for each jaw model in the edentulous part of the jaw. Because of the bone loss it is not possible to place an implant on this part of the jaw,” stated the researchers. “The customized grafts could be manually drawn directly on the surface of the 3D projects by fabricating the original shape of the jaw.”

The void interface set at 0mm between model and graft. The models (n = 6) were evenly divided in two groups, according to the number of missing teeth whether categorized as small defect or large defect. 3D printing was completed on a Form2 3D printer, with the following settings:

“Print resolution was set at 100 microns. To standardize the procedure, every graft and model was printed separately in the center of the build platform with a 135-degree build angle. The print supports were set on the external outline to prevent them interrupting the critical internal area. After printing, the objects were washed twice in two separate 90 percent isopropyl alcohol baths (10 min each) and were postcured for 30 minutes at 45° using a 405nm light box, according to the manufacturer’s protocol.”

The grafts were assessed in vitro, with each one serving as a match for the defect. The researchers state that large-defect grafts did not require any further refining, while small-defect grafts matched after some effort in manually smoothing undercuts. Results of the study were a success, supporting the idea that it is possible to both design and 3D print grafts for alveolar bone augmentation. The researchers state, however, that workflow for this process needs validation in a clinical setting; further, ‘proper vascularization’ must be ensured.

Materials must also be further analyzed for the following:

  • Biocompatibility
  • Osteoconductivity
  • Surface porosity
  • Surface chemistry
  • Tissue bonding
  • Material strength
  • Degradation rate

“The marginal fit of the grafts was better than the internal fit, while the average void dimensions seemed to be correlated to the defect type of the graft. Further in vitro studies with 3D printable bone substitutes are needed for the validation of this digital workflow for alveolar bone augmentation,” concluded the researchers.

While 3D printing has permeated so many complex industrial applications, it may seem surprising to many that it could improve issues having to do with your teeth, mouth, and jaw, also—but just as the technology is becoming more affordable and accessible to others around the world, now so are better dental products and processes, including items like orthodontic aligners, removable partial dentures, and high-performance dental 3D printers available to labs.

Blue and orange colored volume of the void between model and graft.

[Source / Images: ‘Marginal and internal fit of 3D printed resin graft substitutes mimicking alveolar ridge augmentation: An in vitro pilot study’]