Applications of 3D printed bone tissue engineering scaffolds in the stem cell field

2.1. Fused deposition modelling (FDM)

In 1988, Scott Crump, an American scholar, proposed the molding process for the first time. In 1991, Stratasys, Inc. (Eden Prairie, MN, USA) developed the first FDM molding machine and proposed its commercial application. Through the relevant patent search of FDM, it was found that the number of patent applications has been increasing year by year since the technology was proposed in 1988 and reached a peak between 2013 and 2016 (Google patent).

The basic raw materials used in FDM technology are mostly polymer or wax, such as Acrylonitrile Butadiene Styrene plastic (ABS), Polycarbonate (PC), Polyphenylene sulfone resins (PPSU), etc.

As shown in Fig. 1, the working principle of FDM is to transfer the filamentous hot-melt material to the hot-melt printing nozzle through the wire feeding mechanism. The filamentous or linear plastic material is heated to the molten state in the nozzle. Under the control of the computer, the nozzle moves along the shape contour and tracks of the parts, extrudes the molten material, makes it deposit in the expected position and then solidifies. Bonded to the previously formed layers, the layers are stacked to form the product model. Typically, two materials are used during the construction process, one serving as the supporting material and the other constituting the actual building material. Besides, we can change the temperature of the nozzle, the diameter, the moving speed, the extrusion speed and the construction direction to finally influence the porosity, diameter and mechanical properties of the product [21,22].

FDM technology is used to produce customized defect matching structures for bone repair. It not only prints out personalized scaffolds with different porosity and pore sizes to accommodate the growth and differentiation of stem cells [23,24], but also modifies the scaffolds to improve their biocompatibility and bone conductivity [25].

2.2. Extrusion-based 3D printing

Extrusion-based 3D printing, includes a series of techniques: precision extrusion deposition, low temperature deposition modeling, 3D Bio-plotting, etc. [26].

2.3. Stereolithography (SLA)

SLA process was patented in the US by Charles Hull in 1984 and commercialized by 3D Systems, Inc. (USA). Currently, the SLA process is recognized as one of the most deeply researched and the earliest applied 3D printing methods in the world. According to the patent inquiry of SLA technology, it has been found that there were two peaks of patent applications from 2001 to 2004 and from 2013 to 2016 (Google patent).

Photosensitive resin materials mainly include oligomers, reactive diluents and initiators. In 2016 Tethon 3D, Inc. (Nebraska, USA) launched a new ceramic resin for SLA/DLP 3D printers with high-resolution details, thermal shock resistance and thermal and electrical insulation.

As shown in Fig. 3, the technique is based on a photosensitive initiator molecule initiated free radical photo-polymerization. The printed structure is prepared using a photo-curable resin, which is a liquid cured by light irradiation after photo-polymerization. In the SLA devices, the bottom of the structure is formed by polymerization on the top surface of the mobile platform. Thin layers are aggregated by two-dimensional patterns drawn by a guided laser beam. After that, the manufacturing platform descends the patterns on the top of the previous layer polymerization to form the desired structure.

Studies have shown that the controlled macroscopic structure and the microscopic distribution of hydroxyapatite particles produced by SLA technology can enhance the bone promotion effect of composite biomaterials, that is, human bone marrow stem cells can differentiate into the bone in vitro [36]. Also, surface modifications of SLA implants with Sr nano structures have been shown to have a favorable effect on early immunoinflammatory macrophage function and osteoblastic function, leading to enhanced osseointegration outcomes [37]. Studies have shown that the surface of scaffolds treated by cold atmospheric plasma (CAP) is oxygen-rich and the surface roughness is enhanced, which is beneficial to the adhesion, proliferation and chondrogenic differentiation of hMSCs [38].

2.4. Selective laser sintering (SLS)

SLS technology was developed by Carl Dechard at the University of Texas at Austin in 1989. The method was first commercialized by DTM, Inc. (USA), which was later acquired by 3D Systems, Inc [39]. Similar to the previous two technologies, the patent of SLS technology also reached its peak from 2013 to 2016, what worth noting is that 3D companies have the largest share.

In terms of form, the materials used for the SLS process are a wide diversity of powder, including nylon powder, glass powder, wax powder, metal powder, ceramic powder and coated wax metal powder, etc.

As shown in Fig. 4, the powder is supplied in the SLS using powder suppliers and rollers and distributed in thin layers on the build platform. When irradiated with a laser beam, the beam melts the powder in a specified pattern, then lowers the building platform and deposits the fresh powder on top of the molten pattern. Subsequent irradiation of the fresh powder results in the formation of the next layer of the structure. SLS printing is a complex process in which many set values affect the pore size, porosity and mechanical properties of the final product. The main input parameters include layer thickness, scanning speed, laser power, etc. These parameters will affect the energy density of the laser beam in the melted powder layer and finally affect the quality of the final product [40]. Scientists use these differences to meet the needs of different tissue-engineered scaffolds.

Roskies, M et al. used SLS technology to create a customized porous Polyetheretherketone (PEEK) scaffold that maintains the viability of adipose and bone marrow mesenchymal stem cells and induces osteogenic differentiation of adipose-derived mesenchymal stem cells [41]. Different from direct SLS, scientists have explored an indirect SLS technology, in which the traditional raw materials and binders are mixed as the powder raw materials of the SLS machine. After printing, the semi-manufactured scaffolds are sintered at high temperature to remove the binder, and finally the target products are obtained [42,43]. The use of indirect SLS printing can eliminate the shortcomings of conventional direct SLS printing at higher working temperature, that is, the support occurs wavy deformation, hydroxyapatite decomposition [44]. It also showed good mechanical strength, porosity, pore size and promoting osteogenic differentiation [45].

2.5. Printer-based systems

3.1. Joint manufacturing of several 3D printing technologies

Using one 3D printing technology alone may not be able to meet the requirements of the instructions of the 3D printer. For example, in the choice of printing ink, some ink inadequately yields strength to meet the basic requirement of 3D Bio-plotting to print. The scientists solved this problem by using ultraviolet cross-linking from SLA technology, causing that the ink is sheared thin, gelatin-sol transformed, and ultimately achieves excellent printability and fidelity [53,54].

3.2. Combined manufacturing with traditional technology

Although 3D printing technology has a huge advantage over traditional technology, traditional technology is not useless. For instance, scientists used innovative bio-inks to combine FDM technology with casting technology to create new types of osteochondral tissue constructs, producing the scaffolds with excellent mechanical properties and enhanced adhesion, growth, and differentiation of hMSCs [55].

3.3. Direct and indirect manufacturing

In the practical application of 3D printing, researchers have found the limitations of direct printing (e.g. the inability to process low-viscosity materials) and then used indirect printing to solve the problem, and made relevant comparisons [56]. The specific methods are as follows: First, design a negative blueprint of the target build. Sacrifice molds can then be printed through several high-resolution devices to generate a well-defined stand. The material is then cast and cross-linked to form the final shape, and the mold is then removed [57,58]. By using a combination of indirect 3D printing and freeze-drying methods, Bidgoli, M. R. et al., created 3D silk fibroin and silk fibroin-bioactive glass (SF-BG) scaffolds with graded bioactivity to achieve better control, improved uniform pore structure and in vitro bioactivity [59] (see Table 1).

Table 1.

Examples of application.

Technique	Typical resolution	Materials	Cells	Example	Reference
FDM	250–500 μm
	200 μm	nHA, PCL	hMSC		[55]
PED	100–250 μm
	400 μm	PCL, RAD16-I	hMSC		[78]
LDM	250–500 μm
	500–600 μm	BCP, PVA, PRF	BMSCs		[18]
	250 μm	PCL/DCM	MSCs		[80]
3D Bio-plotting	250–400 μm
	25–100 μm	pcHμPs, CHMA, PVA	BMSCs		[54]
	300 μm	PLGA	MSCs		[81]
	100–1000 μm	Alg	ADSC		[82]
	450–1000 μm	Alg	MSCs		[83]
	450–700 μm	PCL/PLGA/Hap	rBMSCs		[84]
	400 μm	MBG/SA–SA	hBMSCs		[75]
	350 μm	Hty	hMSCs		[85]
	400 μm	Alg/gelatin	rBMSC		[86]
	250 μm	PLLA, nHA	MSCs		[13]
	500 μm	printable alginate	SCAPs		[87]
SLA/DLP	15–300 μm
	200 μm	PEG-DA	hMSCs		[17]
SLS/SLM	50–100 μm
	100–300 μm	TiAl6V4	hBMSCs		[88]
	Ra:0.24 ± 0.1 μm	Co–Cr	hADSC		[76]
	400 μm	β-TCP	MSCs		[89]
3D-printer	100 μm
	500–600 μm and 10–50 μm	SF-BG	hBMSCs		[59]
PolyJet	100 μm
		MED610	hMSCs		[52]

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6.1. The advantages of 3D printing

In the past few years, 3D printed bone tissue engineering scaffolds have been increasingly used in the application of stem cells, especially for bone repair and cartilage regeneration of composite structure, which has great advantages that traditional technologies cannot replace. An increasing understanding of the machining parameters that affect these structural properties can help to develop increasingly optimized composites. The 3D printing technology uses many kinds of materials and composite materials, which not only cover the material scope of traditional technology but also derive the material making which could not be realized before by using the technologies (e.g. scaffolds with controllable drug release [75]). 3D printing technology can print highly accurate and complex internal and external structures that adapt to mechanical and biological surface characteristics [76], thus enabling the preparation of customized, patient-specific implants that are highly suitable for tissue and organ defects, as well as disease simulation platforms [77] and stem cell research platforms [78].

6.2. Problems and limitations

In the field of stem cell research and application, 3D printing technology also has disadvantages and limitations. Firstly, although 3D printing can produce personalized and precise scaffolds, it also brings high clinical and scientific research costs and the shortage of mass production. Secondly, some 3D printed scaffold materials are cytotoxic, and the preparation process is also toxic and pathogenic, which also limits its application in clinical, cell biology and regenerative medicine. Besides, there are still huge challenges in generating complex geometric shapes of composite materials, processing various materials and post-optimization of composite surface properties by utilizing the versatility of 3D printing technology. Finally, with the rapid development of stem cell regenerative medicine treatment, the social ethics, legal rationality and regulatory issues brought by printing organs and tissues are also worthy of our consideration and deep thinking [79].

6.3. The future

With the further optimization of current technologies and the emergence of more bio-ink materials, the design of effective scaffolds for the use of extracellular matrix, bone and cartilage for stem cells is becoming more and more promising. 3D printed scaffolds for tissue engineering could be the key to improving the quality of life for patients with organ defects and dysfunction caused by damage or lesions. The development of new printing materials, nanomaterials, especially biocompatible materials, composite materials and complex biomaterials based on various application requirements is the future development direction of 3D printing technology. Also, to promote the systematization, standardization, non-toxic, harmless, green and environmental protection of 3D printing materials, and to continuously expand the integration of 3D printing technology, stem cell technology and traditional treatment will be the development direction of 3D printing and stem cell production. We expect the organic combination of regenerative medicine and additive manufacturing to be a great success for the benefit of all mankind.