Bancroft's Theory and Practice of Histological Techniques

Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion - a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with "built-in" perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.

Organ Printing: Tissue Spheroids as Building Blocks

This review provides a concise summary of the changing phenotypes of macrophages and fibroblastic cells during the local inflammatory response, the onset of tissue repair, and the resolution of inflammation which follow injury to an organ. Both cell populations respond directly to damage and present coordinated sequences of activation states which determine the reparative outcome, ranging from true regeneration of the organ to fibrosis and variable functional deficits. Recent work with mammalian models of organ regeneration, including regeneration of full-thickness skin, hair follicles, ear punch tissues, and digit tips, is summarized and the roles of local immune cells in these systems are discussed. New investigations of the early phase of amphibian limb and tail regeneration, including the effects of pro-inflammatory and anti-inflammatory agents, are then briefly discussed, focusing on the transition from the normally covert inflammatory response to the initiation of the regeneration blastema by migrating fibroblasts and the expression of genes for limb patterning.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/reg2.77

Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration.

Purpose 3D printing technology provides an excellent capability to manufacture customised implants for patients. Now, its applications are also successful in bone tissue engineering. This paper tries to provide a review of the applications of 3D printing in bone tissue engineering. Methods Searching by keywords, from the Scopus database, to identify relevant latest research articles on 3D printing in bone tissue engineering, through “3D printing” “bone tissue engineering”. This study makes a bibliometric analysis of the identified research articles and identified major applications and steps. Results 3D printing technology creates innovative development in bone tissue engineering. It involves the manufacturing of a scaffold with the combination of cells and materials. We identified a total number of 257 research articles through bibliometric analysis by searching through keywords “3D printing” “bone tissue engineering”. This paper studies 3D printing technology and its significant contributions, benefits and steps used for bone tissue engineering. Result discusses the essential elements of bone tissue engineering and identifies its five significant advancements when 3D printing is used. Finally, ten useful applications of 3D printing in bone tissue engineering are identified and studied with a brief description. Conclusion In orthopaedics, bone defects create a high impact on the quality of life of the patient. It leads to a higher demand for bone substitutes for replacement of bone defect. Bone tissue engineering can help to replace a critical defect bone. 3D printing is a useful technology for the fabrication of scaffolds critical in bone tissue engineering. There are different binders which can create bone scaffolds with requisite mechanical strength. These binders are used to create excellent osteoconductive, bioactive scaffolds. Computed tomography (CT) and Magnetic resonance imaging (MRI) help to provide images of specific defects of an individual patient, and these images can further be used for 3D printing the detective object. A bone defect caused by specific disease is sorted out by transplantation in clinical practice. Now a day bone tissue engineering opens a new option for this treatment of bone defects with the manufacturing of porous bone scaffold using 3D printing technology.

3D printing applications in bone tissue engineering.

https://epublications.marquette.edu/cgi/viewcontent.cgi?article=1374&context=dentistry_fac

Applications of 3D printing on craniofacial bone repair: A systematic review.

Three-dimensional (3D) printing is an emerging technology in the field of dentistry. It uses a layer-by-layer manufacturing technique to create scaffolds that can be used for dental tissue engineering applications. While several 3D printing methodologies exist, such as selective laser sintering or fused deposition modeling, this paper will review the applications of 3D printing for craniofacial tissue engineering; in particular for the periodontal complex, dental pulp, alveolar bone, and cartilage. For the periodontal complex, a 3D printed scaffold was attempted to treat a periodontal defect; for dental pulp, hydrogels were created that can support an odontoblastic cell line; for bone and cartilage, a polycaprolactone scaffold with microspheres induced the formation of multiphase fibrocartilaginous tissues. While the current research highlights the development and potential of 3D printing, more research is required to fully understand this technology and for its incorporation into the dental field.

https://www.mdpi.com/2072-666X/10/7/480/pdf

The Applications of 3D Printing for Craniofacial Tissue Engineering.

Objective
Additive manufacturing offers a tailored approach to tissue engineering by providing anatomically precise scaffolds onto which stem cells and growth factors can be supplied. Polyetherketoneketone (PEKK), an ideal candidate biomaterial, is limited by a poor implant–bone interface but can be functionalized with adipose-derived stem cells (ADSC) to promote integration. This in vivo study examined the interaction of a three-dimensional printed PEKK/ADSC implant within the critical-sized mandibular defect in a rabbit model.

Study Design/Methods
Trapezoidal porous scaffolds with dimensions of 1.5 × 1.0 × 0.5 cm were printed using selective laser sintering. ADSCs were seeded on the scaffolds that were then implanted in marginal defects created in New Zealand rabbits. Rabbits were euthanized at 10- and 20-week intervals. Microcomputed tomography was used to characterize bone ingrowth and was correlated with histological analysis. Stress testing was performed on the scaffolds before and after implantation.

Results
All scaffolds were well integrated into adjacent bone. Bone-to-tissue volume increased from 30.34% ( ± 12.46) to 61.27% ( ± 8.24), and trabecular thickness increased from 0.178 mm ( ± 0.069) to 0.331 mm ( ± 0.0306) in the 10- and 20-week groups, respectively, compared to no bone regrowth on the control side (P < 0.05). Histology confirmed integration at the bone–implant interface. Biomechanical testing revealed a compressive resistance 15 times that of bone alone (P < 0.05)

Conclusion
3D-printed PEKK scaffolds combined with ADSCs present a promising solution to improve the bone–implant interface and increase the resistance to forces of mastication after mandibular reconstruction.

Level of Evidence
NA. Laryngoscope, 127:E392–E398, 2017

Three-dimensionally printed polyetherketoneketone scaffolds with mesenchymal stem cells for the reconstruction of critical-sized mandibular defects.

For salivary gland (SG) tissue engineering, we cultured acinar NS-SV-AC cell line or primary SG fibroblasts for 14 days in avian egg yolk plasma (EYP). Media or egg white (EW) supplemented the cultures as they grew in 3D-Cryo histology well inserts. In the second half of this manuscript, we measured EYP’s freeze-thaw gelation and freeze-thaw induced gelled EYP (GEYP), and designed and tested further GEYP tissue engineering applications. With a 3D-Cryo well insert, we tested GEYP as a structural support for 3D cell culture or as a bio-ink for 3D-Bioprinting fluorescent cells. In non-printed EYP + EW or GEYP + EW cultures, sagittal sections of the cultures showed cells remaining above the well’s base. Ki-67 expression was lacking for fibroblasts, contrasting NS-SV-AC’s constant expression. Rheological viscoelastic measurements of GEYP at 37 °C on seven different freezing periods showed constant increase from 0 in mean storage and loss moduli, to 320 Pa and 120 Pa, respectively, after 30 days. We successfully 3D-printed GEYP with controlled geometries. We manually extruded GEYP bio-ink with fluorescence cells into a 3D-Cryo well insert and showed cell positioning. The 3D-Cryo well inserts reveal information on cells in EYP and we demonstrated GEYP cell culture and 3D-printing applications.

https://www.mdpi.com/1996-1944/12/21/3480/pdf

3D Cultures of Salivary Gland Cells in Native or Gelled Egg Yolk Plasma, Combined with Egg White and 3D-Printing of Gelled Egg Yolk Plasma

Objective

A challenge in engineering tissues is to supply parenchymal cells with suitable scaffolds which ideally reproduce the extracellular matrix (ECM). This study tested the hypothesis of preserving the ‘residual connective tissue’ remaining after mechanical and enzymatic release of cells from human submandibular gland biopsies (that we named ‘natural ExtraCellular Matrix scaffolds’, nECMsc) to be used as recycled natural scaffolds. The objective was to test whether nECMsc and native salivary tissue were comparable morphologically, in ECM proteins composition, and in cell seeding efficiency.



Methods

Following cell isolation procedures, nECMsc were kept, either fresh or frozen (sectioned into 12-μm-thick slices), and examined with high-resolution electron microscopy (HRSEM) for its three-dimensional structure, and with picrosirius red staining and immunogold staining for ECM protein composition and distribution, respectively. nECMsc were seeded with human epithelial cells and fibroblasts to assess cell attachment and proliferation in short-term experiments.



Results

Under HRSEM, nECMsc had comparable fiber arrangement to original glands. Histochemical and immunogold-labeling examinations revealed the presence of collagen types I, III, and IV. Seeded epithelial cells and fibroblasts attached, proliferated (14–55%), and were alive (86–99%) after 4–8 days of culture.



Conclusions

nECMsc retained native ECM proteins and maintained their distribution. Seeded cells remained viable on nECMsc.

Natural extracellular matrix scaffolds recycled from human salivary digests: a morphometric study.

Absence of large amounts of orofacial tissues caused by cancerous resections, congenital defects, or trauma results in sequelae such as dysphagia and noticeable scars. Oral-neck tissue regeneration was studied in the axolotl (regenerative amphibian) following a 2.5-mm punch biopsy that simultaneously removed skin, connective tissue, muscle, and cartilage in the tongue and intermandibular region. The untreated wound was studied macroscopically and histologically at 17 different time points ranging from 0 to180 days (N = 120 axolotls). At 12 hr, the wound's surface was smoothened and within 1mm, internal lingual muscular modifications occurred; at the same distance, between days 4-7 lingual muscle degradation was complete. Immunofluorescence indicates complete keratinocytes migration by 48 hr. These cells with epidermal Leydig cells, appearing yellow, lead the chin's deep tissue outgrowth until its closure on the 14th day. Regeneration speeds varied and peaked in time for each tissue, (1) deep chin 84.3 μm/hr from 24 to 96 hr, (2) superficial chin 71.1 μm/hr from 7-14 days, and (3) tongue 86.0 μm/hr between 48 hr and 7 days. Immunofluorescence to Col IV showed basement membrane reconnected between days 30-45 coinciding with the chin's dermal tissue's surface area recovery. New muscle appeared at 21 days and was always preceded by the formation of a collagen bed. Both chin tissues regain all surface area and practically all components while the lingual structure lacks some content but is generally similar to the original. The methodology and high-resolution observations described here are the first of its kind for this animal model and could serve as a basis for future studies in oral and facial regenerative research.

/pdf/oral-facial-tissue-reconstruction-in-the-regenerative-289u7qnu3u.pdf

André M. Charbonneau

Papers

The Applications of 3D Printing for Craniofacial Tissue Engineering.

Three-dimensionally printed polyetherketoneketone scaffolds with mesenchymal stem cells for the reconstruction of critical-sized mandibular defects.

3D Cultures of Salivary Gland Cells in Native or Gelled Egg Yolk Plasma, Combined with Egg White and 3D-Printing of Gelled Egg Yolk Plasma

Natural extracellular matrix scaffolds recycled from human salivary digests: a morphometric study.

Oral-facial tissue reconstruction in the regenerative axolotl