Recent Advances, Challenges and Future Opportunities for the Use of 3D Bioprinting in Large Bone Defect Treatment

Abstract

The healing of bone fractures is a well-known physiological process involving various cell types and signaling molecules interacting at the defect site to repair lost bone tissue. However, large bone defects meaning large tissue loss are a complicated problem in orthopedic surgery. In this chapter, we first present the bone treatment procedure and current commonly employed physical and surgical strategies for the treatment of this kind of fracture such as autografts, allografts, xenografts, and synthetic bone grafts as well as tissue engineering techniques. Further to this, we discuss the common limitations that motivate researchers to develop new strategies to overcome these problems. Finally, we will highlight future prospects and novel technologies such as 3D bioprinting which could overcome some of the mentioned challenges in the field of large bone defect reconstruction, with the benefit of fabricating personalized and vascularized medicine.

1. Introduction

There are 206 bones in human bodies and they consisted of 15% of body weight [1]. Bone defects usually occur by trauma, arthritis, fracture, diseases, etc. and large bone defects need extra intervention to regenerate bone structure and functionality [2]. Bone fractures are very common in societies and around 15.3 million bone fractures with 14 million healthcare visits annually in the United States and it expands with the incensement of the elderly population [3]. The bone defects treatment process can be unsuccessful and lead to delays in treatment, non-unions, and malunions. According to the definition of the Food and Drug Administration (FDA), if the treatment lasts more than 9 months, we are faced with non-unions, but in clinical investigations, this can vary from 2 to 12 months [4]. Considering the high prevalence and the importance of the successfulness of the treatment process, scientists are looking for the best treatment method to treat bone injuries, especially large bone injuries. Today common regeneration replacements are autografts and allografts. Although their limitations such as rare tissue supplements, additional surgeon sites, immunological rejection, and disease transferring restricted their usage. Another treatment method is using implants and bone scaffolds. Although the emergence of tissue engineering in the early 1990s has led to a revolution in bone substitutes, we are facing many challenges in this technique, and efforts must continue to optimize replacement with the highest similarity to natural bone and cartilage tissue [5]. Also, the process of treating bone damage is a complex process that includes various factors. Immune cells and mesenchymal stromal cells (MSCs) cells play an effective role in this process and their behavior should be regulated in such a way that the treatment process is carried out well and does not lead to unwanted reactions such as acute inflammations [6]. Considering the importance of scaffold properties used in cell interactions and as a result in the treatment process, scaffold design should be optimized in such a way that it can provide the desired needs of bone tissue [7]. Recently, 3D printing was introduced as a novel scaffold fabrication method to create a complex cell-laden design with a high mimicking capability of natural tissue using various natural and synthetic materials [8]. Efforts continue to design an ideal scaffold, and perhaps an ideal scaffold for cartilage and bone replacement can be designed in the near future.

In this chapter, the process of bone tissue repair and effective parameters are given first. Then, current used treatment methods and materials for bone tissue repair were summarized. In the following, we will mention the methods of making bone scaffolds based on the 3D printing method and discuss various effective aspects of the 3D printing process, such as the printing methods, the desired needs of the bone scaffolds, and model optimization. Then some of the studies that have been done in this field are discussed. It is hoped that this section can be a guide for researchers in the treatment of bone injuries, especially large bone defects.

2. Regeneration procedure

Replace the entirety of this text with the main body of your chapter. The body is where the author explains experiments, presents and interprets data of one’s research. Authors are free to decide how the main body will be structured. However, you are required to have at least one heading. Please ensure that either British or American English is used consistently in your chapter. Since a complete understanding of the bone healing process will help in finding the best bone replacement, this section describes this procedure. Bone healing procedure is a complex process that involves different cellular and molecular events. The bone healing process can be divided into four main steps: (1) hematoma formation, (2) fibrocartilaginous callus formation, (3) bony callus formation, and (4) bone remodeling [9]. This process requires a systematic order and the failure of any of the steps can lead to the failure of the treatment process. The capability of bone regeneration depends on several factors such as the age of the patient, the condition of the patient, the type of bone, the size of the damaged area, the severity of the damage, blood sources, etc. [10]. The bone healing process involves two main pathways: intramembranous (IM) and endochondral (EC) ossification [11].

In the embryo, the endochondral ossification procedure lead to cartilaginous formation as a template and progressive mineralization in long bones [12]. Therefore, as a result, this process is also effective in-directly in bone repair. After the injury during the endochondral ossification process, inflammatory reactions occur between the hematoma at the end of the fracture and its surrounding environment, which results in the creation of temporary granulation tissue, fibrous tissue, and cartilage, and finally, osteoblasts replace the cartilage tissue leads to bone tissue regeneration [13]. Whereas, in intramembranous ossification, the MSCs directly converted to bone cells without cartilage formation [13, 14].

There are various factors such as cell types, growth factors, cytokines, and chemokines that involves in the bone healing process and can facilitate bone healing. Hematoma is the first step of the healing process. It can act as a temporal natural scaffold and some specific cell types such as macrophages and T cells actives at this level and secretes cytokines and begins the regeneration procedure [15]. The acute inflammatory phase usually accelerates in the first 24 hours and finishes 1 week after injury [16]. Chen et al. [17] mentioned Beta-Catenin as an effective signaling in the healing process of bone injuries. This factor acts differently in various levels of fractures. In the first steps, it is necessary for pluripotent mesenchymal cell differentiation to osteoblasts and chondrocytes and after differentiation, it regulates osteoblasts’ behaviors.

The secretion of several factors involves in this level includes TNF-Alpha, bone morphogenetic proteins (BMP), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, IL-11, and IL-18. The secretion of these factors starts angiogenesis [16]. As the healing process continues, chondrocytes, osteoblasts, and endothelial cells are activated and a cartilaginous callus (soft callus) is created to cover the created gap [18]. The regeneration of large bone defects is challenging in surgery and needs extra attention. Infection followed by long bone implant surgery in femoral, tibial, and humeral fractures is another important issue that should be considered. The treatment is performed according to the infection stage. Stage I infections can be treated by antibiotic administration with/without debridement. In Stage II debridement, IM reaming, antibiotic nails, antibiotics can be useful and the stage III infection is usually treated with debridement, exchange nailing, and systemic antibiotic administration [19]. In fact, large bone defects cannot be treated completely without intervention and need extra medical care [20].

3. Bone healing inhibitors

As mentioned above, the process of bone injury regeneration is a complex process that is affected by various factors. In this section, some of the most important negative influencing factors on the bone healing process are mentioned, which should be controlled as much as possible to improve the healing process.

3.1 Non-steroidal anti-inflammatory drugs (NSAID)

Cyclooxygenase (COX) enzymes classified as COX-1 and COX-2, COX-1 is involved in prostaglandins synthesize in physiological conditions. COX-2 is activated in inflammatory conditions and releases prostaglandins. Whereas, NSAIDs inhibit COX activity [21]. The restriction of prostaglandins synthesized with their critical role in osteoblast and osteoclast functions retarded bone regeneration [22]. However, there is a difference of opinion on the use of NSAID and some studies indicate their positive effects on the treatment process. However, it is better to be careful in using this medicine and limit the duration and dosage [23].

3.2 Smoking

It was reported that smoking and its components significantly affect the fracture union especially the tibial shaft fractures, spinal, and foot and ankle fusions. The smokers expressed a 40% increased time to union compared to non-union smokers [24]. Cigarette smoking contains over 4000 toxins such as nicotine, carbon monoxide, nitrosamines, benzenes, aldehydes, and hydrogen cyanide that can inhibit the proliferation of precursor cells, fibroblasts, red blood cells, and macrophages [25].

3.3 Diabetes mellitus

Diabetes mellitus is the other effective factor in bone healing. Diabetes mellitus affects the skeletal system and increases the chance of osteoporosis and fragility fractures, delays bone healing rates, and reduces bone repair quality [26].

4. Different bone defects treatment techniques

There is no standard for the definition of critical-sized bone defects, however typically a defect greater than 1–2 cm or loss over 50% of the circumference of the bone can be called a critical-sized bone defect. It should be notice that this definition also can be changed according to the defect location site or the situation of surrounding soft tissues [27, 28, 29]. For example femur, 6–15 cm can be classified as a large bone defect due to its good soft-tissue environment, while tibia defects over 1 cm can be defined as a large bone defect due to its lower healing capacity [27]. In another report, bone defects greater than 3, 5, and 6 cm in the forearm, femur/tibia, and humerus, respectively, were considered critical-sized defects [30]. Therefore, various parameters should be considered in the critical-sized bone defect and in general, it can be defined as an osseous defect that cannot be healed spontaneously during the lifetime or less than 10% of bone regenerated during the lifetime [31]. Different treatment methods have been reported for the treatment of bone complications, especially large bone defects, some of the most important treatment methods are summarized below. Table 1 summarized the mentioned methods with their advantages and disadvantages.

Treatment method	Categorize	Description	Defect size treatment	Advantages	Disadvantages
Electrical stimulation	Physical treatments	Applying Electrical stimulation to enhance cells differentiation	Low	It can be non-invasive, stimulation of cells proliferation and differentiation	It can be invasive
Low-Intensity pulsed ultrasonography		Applying ultrasound to stimulate cells	Low	Non-invasive	low effectiveness on the pain reduction, poor functional recovery, and poor capability to reduce the recurrent fracture
Extracorporeal shock wave therapy		Applying sequence of high energy acoustic impulses to stimulate cells	Low	Non-invasive, safe, easy, can be applied on most patients	Low efficiency
Ilizarov method	Surgical techniques	applying compression or distraction forces to the bone defects	<9.5 cm [32]	simple, effective, common, minimally invasive	pin tract infection, unconnected bone, re-fracture, bone malformation and transform death
Masquelet -induced membrane		A two-step treatment method with the aim of the soft tissue formation inhibition in the defect site in the first step and bone regeneration in the second step	<25 cm [33]	simple, lower rate of infections	Loosening of the fixation implant, fracture through the graft,and bone resorption.
Autograft	Grafts	a graft of tissue for another site of the same individual’s body	<5 cm [34]	High osteoinductive and osteoconductive properties, high success rate, and low probability of immune rejection	Limitation of harvest and formation of secondary surgeon site, fast absorption of autologous
Allograft		non-self-human graft	<3 cm [35]	Providing natural bone structure	risk of disease transmission (HIV, hepatitis B, hepatitis C, and other viruses), and rareness of supplements
Bone morphogenetic proteins (BMPs)	bioactive molecules, and growth factors	It is a transforming growth factor beta for bone regeneration	<7 cm [36]	high osteogenic potential, FDA approve	side effects such as ectopic osteogenesis, osteolysis, bone cysts, local inflammation, traumatic injuries, postoperative fever, and hemorrhage, or even cancers in excess administration
Statins		3-hydroxy-3-methylglutaryl-coenzyme-A	n.i*	Regulating BMP-2 gene expression and show an osteoinductive role	local retention of the statin
Fibroblast growth factor-2		a famous family member of fibroblast factors	n.i	accelerate cell proliferation, collagen synthesis enhancer, angiogenesis, and osteogenesis	short half-life (3–10 min)
Platelet-Rich Plasma		Using a high concentration of platelets with a lot of therapeutic applications	Cannot be effective in 2.5 cm bone defects [37]	Releasing different growth factors with high regeneration potential	higher infection rate
Insulin-like growth factors		one of the most abundant growth factors in bone matrix	n.i	osteoblasts growth and accelerates bone healing	Not reported
Bisphosphonates		drugs with inhibitory effects on the osteoclasts	n.i	Decrease bone resorption	Increase the risk of fractures and esophageal cancer
dECM		Decellularized natural ECM	n.i	Providing natural ECM structures containing the bone components such as growth factors and cytokines	The possibility of disease transmission
Hydroxyapatite		The main inorganic phase of bones	<10 cm [38]; ≤15 cm^3 [39]	Providing the natural bone component	low dispersibility and tendency to form aggregates
Cements	Cements	Injectable mixing biomaterial powder with a liquid that can be set in the defect site	<25 cm [40]	Filling complex defects shapes, injectability	thermal necrosis of the bone, cement fragmentation, and foreign body reaction to wear debris
Combination	Combination	Simultaneous usage of different treatment techniques	It depends on the applied techniques	Synergic effects of the used techniques and overcome each other’s limitations	It depends on the applied techniques

Table 1.

Comparison of treatment methods for bone injuries along with advantages and disadvantages.

*

No identified range (n.i).

4.1 Physical treatments

4.1.1 Electrical stimulation

Electrical stimulation can enhance MSCs’ adhesion, alignment, division, proliferation, migration, and differentiation [41, 42]. Electrical stimulation can be applied invasive, semi-invasive, or non-invasively. In the invasive method, the DC stimulator electrodes are implanted in the bone injuries and will remove at the end of the regeneration procedure. In the semi-invasive method, the cathodes were placed percutaneously. In the non-invasive method, stimulators were inserted on the skin [43]. Two main mechanisms possibly direct the response of the cells to electrical fields. The first mechanism includes the downstream activation as a consequence of asymmetric redistribution/diffusion of electrically charged surface cell receptors and the other mechanism is attributed to the voltage-gated Ca²⁺ channels activation and the following cellular responses [42].

4.1.2 Low-intensity pulsed ultrasonography

Ultrasound treatment is a safe non-invasive method and it was started in the early 1930s for therapy applications [44]. For bone healing Low-Intensity pulsed ultrasonography, the ultrasound is applied every day at 1.5 MHz, with 1 kHz pulse and 30 mW/cm² for 20 min time periods [45]. Both bone-forming osteoblasts and mechano-sensitive osteocyte cells can sense the applied ultrasound. Osteoblast accelerates bone formation while regulating inflammatory responses. On the other hand, osteocytes that consisted high percentage of bone cells can sense acoustic radiation force created by ultrasound which leads to biological signals and enhance bone formation [46]. However, some limitation was detected in Low-Intensity pulsed ultrasonography such as low effect on pain reduction, poor functional recovery, and poor capability to reduce recurrent fracture [44].

4.1.3 Extracorporeal shock wave therapy

Extracorporeal shock wave therapy is a non-invasive therapy technique and can be defined as a sequence of high-energy acoustic impulses with a different course that can produce pressure changes while it propagates [47]. This technique has some advantages such as being safe, non-invasive, easy, and can apply to most patients, however, some surgeons believed that it has no significant difference from a placebo [48]. While it was demonstrated that extracorporeal shock wave therapy can be involved in the activation of various biochemical signals that trigger neoangiogenesis, fibroblast proliferation, collagen synthesis, and finally tissue regeneration. On the other hand, the negative phase of the shocks can enhance cell membrane permeability and inhibit calcification deposits and reduce pain [49]. The involved cellular signaling pathways during extracorporeal shock wave therapy are focal adhesion kinase, Extracellular-signal-regulated kinase, Wnt/β-catenin, Protein kinase R-like endoplasmic reticulum kinase/activated transcription factor, ATP/P2X7, Brain-derived neurotrophic factor [50].

4.2 Surgical techniques

Ilizarov method is a simple, effective, common, minimally invasive external fixation technique based on applying compression or distraction forces to the bone defects due to osteogenesis induced by tension stress [51]. Although the Ilizarov method is an applicable technique, especially for large bone defects, in some cases it may be associated with dissatisfaction such as pin tract infection, unconnected bone, re-fracture, bone malformation and transform death reported [52, 53]. One of the other popular surgical methods especially in large bone defects is Masquelet induced membrane [54]. This procedure includes two main steps: first, the created cavity is carefully debridement then is filled with polymethyl methacrylate cement to produce a membrane and inhibit soft tissue formation. In the second step, the cement is removed and the iliac crest cancellous bone or RIA (Reamer-Irrigator-Aspirator) cancellous bone is used to fill the cavity [54, 55].

4.3 Using grafts, bioactive molecules, growth factors, and cement for bone regeneration

4.3.1 Autograft

The gold standard and first bone replacements is autologous bone due to its high osteoinductive and osteoconductive properties [56]. The autografts contain natural bone contents such as bone cells, growth factors, and natural extracellular matrix components such as collagen, non-collagenous proteins, and hydroxyapatite [56]. Although this method has good benefits, it also comes with challenges, for example, a secondary operation that causes suffering for the patient or limitations in the dimensions of the harvested bone, and fast absorption of autologous implant that should be regulated [57].

4.3.2 Allograft

Allografts are non-self-human that can be used as the other option for use in bone defects treatment and consist of one-third of all-bone grafts in North America [58]. The osteoinductivity and mechanical properties of the allograft can be reduced during the freezing, and sterilization processes, while the ECM can provide a natural scaffold for bone regeneration [59]. The main restriction of allografts is the risk of disease transmission (HIV, hepatitis B, hepatitis C, and other viruses), and the rareness of supplements [60].

4.3.3 Bone morphogenetic proteins (BMPs)

BMPs are the most popular subfamily of transforming growth factor beta that are necessary for fetal tissue development and fracture healing [61]. Bone morphogenetic protein-2 (BMP-2) shows high osteogenic potential and can be applied in bone regeneration [62]. It is the only osteoinductive growth factor with FDA approval for clinical usage, but its administration should be controlled. Excess administrated BMPs, especially in the first levels of bone mineralization can cause different side effects such as ectopic osteogenesis, osteolysis, bone cysts, local inflammation, traumatic injuries, postoperative fever, hemorrhage, or even cancers [63].

4.3.4 Statins

Statins, 3-hydroxy-3-methylglutaryl-coenzyme-A, are a family class of drugs reductase inhibitors, with impressive impact on the bone healing procedure. This can be due to the BMP-2 bone factor induction that can facilitate bone healing [64]. The most famous member of this group for bone regeneration applications is Simvastatin [65]. Simvastatin can accelerate bone regeneration by regulating BMP-2 gene expression and show an osteoinductive role through the Ras signaling pathway, and activate mitogen-activated protein kinases. Also, it can inhibit bone density reduction using estrogen receptor-alpha or BMP-2 induction [66, 67]. Feng et al. [68] demonstrated the capability of simvastatin to enhance osteoblast differentiation of mesenchymal stem cells in osteoporosis Sprague–Dawley rats. In another study, Shahrezaee et al. [69] investigated and compared the systemic delivery of atorvastatin, simvastatin, and lovastatin on bone healing. For this purpose, the drugs were orally administrated to the thirty ovariectomized female Sprague–Dawley rats, and the results were analyzed after 60 days. According to the achievements, atorvastatin had no significant effect on bone density, while simvastatin and lovastatin enhanced calcium levels, osteogenic gene expression, bone density, and biomechanical properties. The CT scan of different analyzed groups is shown in Figure 1 (A-E) and can prove the better function of simvastatin and lovastatin. In comparison, the simvastatin-treated sample showed a significantly higher resemblance to normal groups and demonstrated increased osteogenic genes and better biomechanical performance.

Figure 1.

(A-E): Micro CT results of different analyzed groups after 60 days: (a): Normal, (B): Negative control, (C): Simvastatin, (D): Lovastatin, (E): Atorvastatin [69]. (F-K): The histological investigation of three control (F), sham (G), and experimental (H) groups after 1 week and control (I), sham (J), and experimental (K) groups after 8 weeks. The bottom images are the higher magnifications: (I1) fibrous connective tissue of control, (I2) control scaffolds covered with fibrous, (J1) fibrous connective tissue of sham, (J2) sham scaffolds covered with mixed tissue, (K1) cartilage and bone tissue of experimental group, (K2) experimental group covered with cartilage and bone tissue [70].

4.3.5 Fibroblast growth factor-2

Fibroblast growth factor-2 is a famous family member of fibroblast factors with unique properties such as mitotic promoters that can accelerate cell proliferation, collagen synthesis enhancer, angiogenesis, and osteogenesis [71]. However, its short half-life (3–10 min) should be regulated using different drug release carriers because excess Fibroblast growth factor-2 usage can cause side effects and depressant bone regeneration [72].

4.3.6 Platelet-rich plasma

Platelet-Rich Plasma (PRP) refers to the high concentration of platelets (two to five higher than blood) in a small volume of plasma with a lot of therapeutic applications due to the capability of platelets to secret different growth factors [73]. It was reported that approximately 300 bioactive cytokines exist in PRP and their release can trigger various cellular interactions and facilitate tissue regeneration. Although there is no international protocol for PRP usage [74]. The main growth factors are platelet-derived growth factor (PDGFaa, PDGFbb, PDGFab), TGFβ1/TGFβ2, VEGF, FGF, epithelial growth factor (EGF), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF) and insulin-like growth factor (IGF) [75]. These growth factors can modulate inflammation, accelerate cell proliferation and differentiation, promote angiogenesis, and as a result accelerate bone regeneration [76]. The performance of PRP in large bone treatment is successful with a lower rate of pain score and shorter healing duration. However, a higher infection rate was reported [77].

4.3.7 Insulin-like growth factors

Insulin-like growth factor-1 (IGF-1) is one of the most abundant growth factors in bone matrix with a wide range of physiological functions [78]. The administration of IGF-1 lead to enhance osteoblasts growth and accelerate bone healing [79]. IGF receptor is a cell surface receptor named an Insulin-like growth factor 1 receptor and after binding to Insulin-like growth factors, it activates two main signaling pathways: the phosphoinositide 3-kinase (PI3 K)/AKT and the RAS/mitogen-activated protein kinase (MAPK) and leads to promoting cell proliferation, migration, and proliferation [80]. In addition to IGF-1, metformin can improve osteogenic differentiation and facilitate bone regeneration [81]. Shahrezaei et al. [82] reported that metformin coating of poly (lactic acid) and polycaprolactone scaffold increased the osteogenic and angiogenic markers expressions and accelerated the regeneration of rats’ large bone defects.

4.3.8 Bisphosphonates

Bisphosphonates are drugs with inhibitory effects on the osteoclasts using increasing their apoptosis rate of them and interfering with resorption signals sent from the bone matrix [83]. These drugs are classified into two main categories with different mechanisms: nitrogen-containing bisphosphonates, and non-nitrogen-containing bisphosphonates. The non-nitrogen ones metabolized to analogs of adenosine triphosphate and interfere with mitochondrial activity and lead to cell death, while the nitrogen-containing ones inhibit farnesyl pyrophosphate synthase which can be effective in protein transportation and lead to cell death and inhibit osteoclast maturation [84].

4.3.9 dECM

The extracellular matrix (ECM) is a complex structure with various components including collagen, glycosaminoglycans, chondroitin sulfate, elastin, etc. [85]. In the decellularization procedure of ECM, the cells were eliminated with low harm to the natural structure and components of the ECM [85, 86]. Decellularized extracellular matrix (dECM) derived from natural ECM can be used as a biomaterial in tissue engineering applications [87]. The dECM introduces a natural structure for bone replacement containing the bone components such as growth factors and cytokines [88]. Also, dECM can provide cellular activities that are necessary for bone healing [89].

4.3.10 Hydroxyapatite

Natural bone ECM consists of organic and inorganic phases and the base of the organic phase is calcium phosphate, especially hydroxyapatite [90]. Therefore, hydroxyapatite is one of the most studied materials in bone regeneration applications [91, 92, 93, 94]. The hydroxyapatite can be used alone, synthetically, porously, in a resorbable form, or combined with natural or synthetic materials such as β-TCP, PRP, collagen, and bioactive glass [95]. Walsh et al. [96] showed a high potential of hydroxyapatite-collagen scaffold for the treatment of critical-sized bone defects with 28-fold and 7-fold increments in the bone volume and new bone area after 4 weeks.

4.3.11 Cements

Cement is used clinically due to its injection capability and molding as a paste and filling the irregular bone defects in a minimally invasive manner [97]. The Cement is obtained by mixing biomaterial powder with a liquid that can be set when injected into the defect site [98]. Polymethyl methacrylate is one of the common cement to repair bone defects [99]. Jia et al. [100] demonstrated the positive application of antibiotic PMMA cement (0.5 g of gentamicin per 40 g of PMMA) coating on the locking plates for infection inhabitation and enhancing bone regeneration. On the other hand, self-setting calcium phosphate is the other common type of cement with a lot of applications. These cements provide injectibility with favorable bone defect-filling capability [101].

4.4 Combining techniques

Although each of the treatment methods mentioned above has benefits and potential effects, however, all methods face limitations. The simultaneous use and combination of the mentioned methods to provide a new treatment method can lead to the simultaneous use of the benefits of each method in such a way that they overcome each other’s limitations. Leppik et al. [70] proposed a combined technique for the treatment of large bone defects by using tissue engineering and electrical stimulation. For this purpose, the test was analyzed in-vitro and in-vivo. The in-vivo experiment was performed on three different groups: large femur-defected rats treated with β -TCP scaffolds (control group), β-TCP scaffolds and Adipose tissue-derived mesenchymal stem cells (AT-MSC) (sham group), electrical stimulation, β-TCP scaffolds, and Adipose tissue-derived mesenchymal stem cells (experimental group). The 3 cm defects were created in the right limbs of the rats under general anesthesia. After 1 week, Staining was performed using Alcian Blue, Orange-G, and Hematoxylin histological investigation was carried out (Figure 1 (F-K)), the 80% defect gap was covered with soft tissue in the experimental group, while there was a high uncovered gap was seen in the control and sham groups. Although there can be a better condition in the sham group in comparison with control samples. After 8 weeks, histological observations showed that cartilage and bone tissue were formed properly in the experimental group.

In another combined treatment method, Qi et al. [78] used Electrical stimulation and insulin-like growth factor-1 (IGF-1) for bone regeneration. The effect of this method was tested on MC3T3-E1 cell behavior and the results were analyzed with different techniques. According to the achievements, the combination of electrical stimulation and IGF-1 acted synergistically and lead to higher expression of osteogenic marker genes and alkaline phosphatase activity, and cell mineralization. The results of alizarin red staining of the cell mineralization were shown in Figure 2 (A-H). The combination techniques demonstrated higher calcium content in comparison with the cells that only receive the IGF-1 factor. On the other hand, when the concentration of IGF-1 was 100 and 200 ng/ml a higher calcium content was detected. In another clinical trial, Combal et al. [102] combined the Masquelet-Induced Membrane and Capanna Vascularized Fibula with an Allograft large femoral bone defects and introduced it as Capasquelet. The technique can be used for the treatment of bone defects with at least 10 cm loss. For this purpose, four patients suffering from tumors/traumatic bone defects were operated on with this combination technique. The result proved the important effect of the induced membrane in the regeneration process with fast allograft and fibula union and early weight-bearing Figure 2 (I-J).

Figure 2.

(A-H): Alizarin red staining results of the calcium content after 21 days of electrical stimulation and IGF-1 factor treatment (a). The samples are (B) control, (C) IGF (50 ng/ml), (D) IGF (100 ng/ml), (E) IGF (200 ng/ml), (F) ES + IGF (50 ng/ml), (G) ES + IGF (100 ng/ml), and (H) ES + IGF (200 ng/ml) [78]. (I-J): Control X-rays for patient No. 1 (I) 3 months (J) 10 months postoperatively [102].

4.5 Novel techniques

4.5.1 Cell therapy

Few cell types can form heterotrophic bones while many cell types can create mineralized tissues [103]. Progenitor cells are the best cell population for cell therapy applications. The number of the needed cells depends on different factors such as bone fracture types and size, cell source, therapeutic method, and biomaterials, whereas usually for a 4 cm large bone defect approximately 600 million cells are sufficient [104]. Mesenchymal stem cells are extensively studied in bone regeneration with favorable clinical application, especially in dental and orthopedic fields [105]. Furthermore, MSCs can regulate physical contact between cells and enhance cell–cell interactions for osteogenesis [106]. Although these cells can be isolated from different parts of the body, the gold standard is included bone marrow-derived MSCs (BM-MSCs) due to their easiness and high osteogenic capacity [107]. BMSCs are the most popular cell population for bone treatment over 50 years ago [104]. In the Hernigou process, the bone marrow aspirates to the bone defect sites [106]. Furthermore, the infusion of BMSCs is used for the treatment of children with severe osteogenesis imperfect [108].

4.5.2 Tissue engineering

The limitations of the previous bone replacements such as donor-site scarcity, high risk of disease transmission, and immune rejection made the scientists find a new solution. Tissue engineering introduced promising strategies for bone regeneration [109, 110, 111].

Tissue engineering consisted of cells, scaffolds, and bioactive molecules. Both natural and synthetic materials can be used for scaffold fabrication in tissue engineering with their unique properties. Natural materials provide better biological recognition and cellular interactions [112]. Synthetic materials can be modified according to the requirements and the source is usually abundant. On the other hand, the material should be modified to overcome usual synthetic materials disadvantages such as low material-tissue integrity, low vascularization, poor cell attachment, and therefore functional bone regeneration [113, 114].

5. Specific bone scaffolds requirements

The scaffold is one of the key elements of tissue engineering that provide mechanical support for cell adhesion and growth [115]. To design an ideal scaffold material selection, scaffold architecture, and scaffold fabrication techniques create important roles [116]. Bone scaffolds should provide a high porosity for cell adhesion, proliferation, and growth; meanwhile providing adequate mechanical properties during bone regeneration [117, 118]. It was suggested 75–95 vol% porosity is proper for bone scaffolds [119]. The osteoblast cells are in the range of 10–50 μm and prefer the larger pore size (100–200 μm) for growth and mineralization that could allow the penetration of macrophages, cell migration, and vascularization. Whereas lower pore size of this range leads to the creation of fibrous tissue and inhibits bone tissue regeneration [120].

The other important parameter for designing scaffolds is the swelling ratio. There are various properties affecting the swelling ratio such as hydrophilicity, cross-link density, porosity, pH, and the environmental characteristics [121, 122, 123, 124]. A high swelling ratio facilitates fluid penetration and as a result enhances nutrition, oxygen, and waste material transportation, while the structures with an excess swelling ratio usually do not show adequate mechanical properties for bone healing [125]. The range of mechanical properties depends on the bone type, however, in general for elastic modulus range is reported 18.6 ± 28.8 GPa and 10–157 MPa for cortical and cancellous bone, respectively [126]. Therefore, it is expected the designed scaffold could provide sufficient mechanical properties for bone tissue during the regeneration procedure. Furthermore, the biodegradation ratio of the construct scaffolds should match the tissue growth rate [127]. Each bone requires a specific degradation ratio for example in spinal fusion regeneration it needs 9 months while it is suggested 3–6 months for craniomaxillofacial healing [119].

6. Scaffolds fabrication methods

Scaffolds act as a temporal template for cell attachments and growth that can contain different cell types, growth factors, drugs, and bioactive molecules. Although the material selection had a great impact on the scaffolds’ properties, the scaffold’s fabrication technique is also an important criterion. There are different techniques for scaffold fabrication such as gas-foaming, phase separation, particle leaching, electrospinning, freeze-drying, freeze-casting, and 3D bio-printing. An ideal scaffold should be able to create the most similar structure to the natural bone tissue along the same physical, chemical, and biological properties while providing reproducibility with minimal cost and time. The conventional scaffold fabrication methods showed poor controllability during the creation process to fabricate complex structures with tunable micro-scale and macro-scale. The recent scaffold fabrication methods such as 3D bio-printing allow high controllability, the capability of complex structures, and the inclusion of the cells and growth factors in the structure [128, 129, 130].

6.1 Three dimensional bio-printing

Recently, 3D bio-printing is introduced as a novel scaffold fabrication method and has provided numerous capabilities compared to the traditional methods such as a lower rate of residual organic solvents which decreases the harmful effects of these chemical solvents on the cell’s survival and interactions, or scaffold designing before printing which leads to personalization [131, 132]. Also, it is possible to build complex structures with/without cells [133]. The most important benefit of 3D bio-printing for bone tissue engineering is controlling cell distribution [134]. The printing materials that contain cells are called ‘bio-ink’ and should demonstrate some features for cell survival and the creation of a favorable bone substitute. The most important features in the bio-inks selection are biocompatibility, printability, fidelity, viscoelasticity, shear-thinning, yield stress, shelf life, cross-linking capability, and even cost [135].

6.1.1 Methods

In the first years of the emergence of 3D printing, conventional methods of this technique such as fused deposition modeling (FDM), stereolithography (SLA), and selective laser sintering (SLS) were used to print tissue engineering structures. Although these methods provided unique capabilities compared to the traditional scaffold construction methods, however, as time passed and the disadvantages of these methods became clear, such as the inability to load cells and bioactive molecules, the inadequate resolution, and the impossibility of using a wide range of materials led researchers to develop 3D bio-printing technology. In recent years, the use of 3D bio-printing technology has accelerated. This method is divided into three main types: inkjet-based bioprinting, extrusion-based bioprinting, and laser-assisted bioprinting [136, 137, 138, 139]. Inkjet-based bioprinting includes the deposition of bio-ink droplets onto a substrate. Although this method can print a cell-laden scaffold at a high speed, the number of biomaterials that can be used in an inkjet printer is limited, due to the rapid gelation time requirement. The extrusion-based bioprinting method is extruding high cell density laden bio-inks to create scaffold structures. It is the most easiest and popular 3D bio-printing method. In laser-assisted bioprinting, the bio-inks are printed using the laser in a nozzle-free manner with higher resolution. The laser heats the determined points and deposits the cells in the exact place [140].

6.1.2 Bone model optimizing

As mentioned before, the 3D bio-printing can create personalized complex structures. The computer modeling of the 3D bio-printing structures has two main steps. The first step is data acquisition using computed tomography (CT) or magnetic resonance imaging (MRI) and the second step is image processing and model generation [141]. The 3D models should be predictable and shows controllable shape change. Therefore, the 3D model design should be optimized by considering chemical composition, volume fraction, and structural features such as pore size, pore shape, pore distributions, and mechanical properties [142]. The finite element analysis can be used to calculate the mechanical properties of the designed structures and predict the results, theoretically. Liang et al. [143] demonstrated the tetragonal structures showed better mechanics than the tetragonal, hexagonal, and wheel-like designed structures for bone replacements using finite element analysis and an electromechanical universal testing system. The combination of state-of-the-art optimization techniques with computer models can provide a basis for predicting scaffold behavior and optimizing the designed scaffolds for bone regeneration, especially for large bone defect regeneration [144].

7. The previous studies on large bone treatment using 3D bio-printing

Pitaco et al. [145] developed a hypertrophic cartilage graft for the replacement of critical-sized defects. The bio-ink was prepared with Human mesenchymal stem/stromal cells loading in the fibrin-based bio-ink and 3D bio-printing was performed in the polycaprolactone framework. The designed constructs were implanted into nude mice and expressed a high potential for bone remodeling with high efficiency in vascularization and bone formation. In another trial Sun et al. [146] introduced a multi-component bio-ink for the treatment of critical-sized bone defects in diabetes mellitus patients. The gelatin, gelatin methacryloyl, and 4-arm poly (ethylene glycol) acrylate bio-ink were loaded with BMSCs, RAW264.7 macrophages, and BMP-4-loaded mesoporous silica nanoparticles. Silica nanoparticles could enhance the mechanical stability and control the release of BMP-4. The existence of BMP-4 led to the polarization of RAW264.7 to M2 macrophages to produce BMP-2 and anti-inflammatory factors and the dual presence of BMP-2 and BMP-4 facilitated the osteogenic differentiation of BMSCs. Zhang et al. [147] developed a human mesenchymal stem cell-laden graphene oxide/alginate/gelatin bio-ink with different concentrations of graphene oxide (0.5, 1, and 2 mg/ml) to create bone scaffolds. The results demonstrated that the scaffolds containing 1 mg/ml graphene oxide had higher shape fidelity, osteogenic differentiation, and mineral volume for mimicking critical-sized calvarial bone defects. Furthermore, Dong et al. [148] evaluated the rabbit bone marrow mesenchymal stem cells and bone morphogenetic protein-2 encapsulated chitosan hydrogel in 3D printed polycaprolactone scaffolds with the aim of bone tissue regeneration. The cellular investigation after 2 weeks confirmed high osteogenesis and bone matrix formation of the proposed scaffold. Khoshnood et al. [149] introduced a novel 3D bio-printed composition for bone regeneration. According to their study, printing 1 wt % tragacanth and 3 wt % alginate bio-ink can provide favorable printability and viscosity. The addition of tragacanth to the alginate modulates the biodegradation ratio to 21 days. Furthermore, adding hydroxyapatite improved osteoconductivity. The prepared bio-inks provided high cell differentiation support with the expression of CD105, CD44, and CD90 surface markers. Hao et al. [150] clinically investigated the printing capacity to treat complicated and large acetabular bone defects. The personalized titanium prosthesis was designed based on computed tomography and X-ray and the final model was achieved using Siemens NX software and Magics software and then the printing was performed. After this step, the prosthesis was trimmed, polished, sandblasted, and cleaned. Then, a surgical process was performed. According to the radiographic and software analysis, the 3D-printed prosthesis showed a high Harris hip score recovery rate in all patients. Wang et al. [151] used 3D-printed polylactic acid (PLA) and nano-hydroxyapatite scaffolds for large bone treatment. The polymeric part was used to provide the required mechanical and degradation rate of the bone scaffolds and nano-hydroxyapatite was added to provide osteoconductivity and improve the load-bearing capacity. The prepared scaffolds were tested in-vivo in the rabbit model. The high biocompatibility and osteogenic properties proved the high potential of these scaffolds for further future studies. Li et al. [152] introduced bio-active 3D printed scaffolds for the regeneration of large and load-bearing bone defects. The scaffolds compound was strontium-hardystonite-gahnite and the scaffolds were tested in the large bone-sized defects of the sheep tibia for 3 and 12 months. Mathematical modeling confirmed and highlighted the importance of the surgical process and implant fixation as much as the implant compositions and the in-vivo results revealed high induction of bone formation after 12 months.

7.1 In-situ printing

In-situ printing or in-vivo 3D bio-printing is defined as the direct printing of bio-inks at the defects’ site with the aim of creating or repairing damaged tissue in a clinical setting. In this procedure, the robotic arms are assembled with the bio-printing unit, and the printing is conducted in minimally invasive routes in the defect site [153]. Cohen et al. [154] used in-situ bio-printing for the treatment of osteochondral defects. The alginate and demineralized bone matrix were printed using a built-in-house extrusion-based bio-printer for this purpose. Two types of defects were created in the calf femur: a cartilage only and an osteochondral defect. The results demonstrated the high geometric precision of the in-situ printing process. In another study, Kerique et al. [155] in situ printed the Nano-hydroxyapatite in the mouse calvaria defect model using a laser printer. They suggested that the in-situ printing usage by surgeons or actual robots can reduce the used materials to pL volume and the spatial resolution increased. In another trial on the robotic in-situ bio-printing for large bone healing, Li et al. [156] presented a new method for optimizing robotic printing. The extrusion-based bio-printer was selected due to the easier structural and mechanical controllability and a modified 4-DOF robot with higher kinematic accuracy of 0.5 mm was used. The bio-printing was performed using a double-network hydrogel bio-ink consisting of alginate, polyethene glycol diacrylate (PEGDA), and methacrylated gelatin (GelMA). Alginate was cross-linked with calcium ions and PEGDA and GelMA created a covalently cross-linked network. The in-vivo successful results on the large tibia bone defects of pigs revealed the high potential of this method for future clinical applications of in-situ large bone regeneration.

7.2 Pre-vascular scaffolds

Angiogenesis is an important factor in the bone healing process in both intramembranous and endochondral ossification healing ways [157]. In endochondral ossification cartilaginous continues in the vascular network and the network provides cell sources, nutrition, oxygen, and growth factors needed for the repair process, and then osseous tissue formed [18]. Osteogenesis is closely related to angiogenesis during neo-bone tissue formation [158]. Therefore, efforts to create pre-vascular structures in order to create fully functional and perfusable tissue constructs for clinical applications have been made with the help of 3D bio-printing [159]. Kuss et al. [160] designed a pre-vascularized scaffold with the co-culture of Human adipose-derived mesenchymal stem cells (ADMSC) and human umbilical vein endothelial cells (HUVEC) on the polycaprolactone/hydroxyapatite scaffolds. Then, the pre-vascularized scaffolds were implanted in the nude mice, and vascularization capacity was analyzed. The results confirmed the microvessel and lumen formation and increased the vascular network formation. In addition, Nulty et al. [161] implanted the prevascularized 3D-printed polycaprolactone scaffolds to treat critical-size bone defects. The prevascularization was performed using both human bone marrow stem/stromal cells (hBMSCs) and human umbilical vein endothelial cells (HUVEC) in a fibrin base bio-ink. The in-vivo results showed increased vascularization and as a result bone regeneration.

7.3 Osteochondral scaffolds

Osteoarthritis is the most prevalent joint disease that affects millions of people around the world. The growing elderly population increases its prevalence and needs extra attention [162]. The avascular nature and rare source of repair cells lead to the restricted self-regeneration capability of cartilage [163] and existing treatment methods mostly repair single cartilage tissue and are not suitable for the whole osteochondral replacement [164]. As a result, the search for the best osteochondral substitute continues.

A study by Gardner et al. [165] investigated the composition and the porosity of the clinically available polycaprolactone (PCL) and β -tricalcium phosphate (β-TCP) and proposed the selected scaffold for intervertebral disc replacement. The results demonstrated that β-TCP facilitated the proliferation and differentiation of the C3H10 cells. According to the results, the design consisted of 45% porous material with 60% of β-TCP for the topper and lower parts and the core consisted of polycaprolactone with 70–75% porosity to stimulate the cartilage section of the intervertebral disc (Figure 3 (A-B)). In another study, Kilian et al. [166] introduced a novel combination for osteochondral bio-printing. The bio-ink included the alginate-methylcellulose with calcium phosphate cement. The multi-channel 3D plotted structure consisted of three parts. The upper part contained the hMSC cell-laden hydrogel and partly mineralized calcium phosphate cement. The middle part was a biphasic interwoven network of both hMSC cell-laden hydrogel and calcium phosphate cement to mimic calcified cartilage and the bottom part was the subchondral bone that was created using pure calcium phosphate cement (Figure 3 (C)). The results demonstrated high efficiency of cell survival with the potential of redifferentiation to produce cartilage extracellular matrix (ECM) components.

Figure 3.

(A-B): Novel 3D printed polycaprolactone and b-tricalcium phosphate (β-TCP) scaffold for intervertebral disc replacement [165]. (C) the multi-component 3D plotted scaffolds designed by Kilian et al. [166] for osteochondral treatment consisted of three main parts to stimulate natural osteochondral structures.

Shim et al. [167] described a 3D bio-printed construct with different ECM types for osteochondral tissue engineering. The bio-ink was constructed on the base of atelocollagen and supramolecular hyaluronic acid loaded with human mesenchymal stromal cells. The mixture of Cucurbit[6]uril/conjugated HA and 1,6-diaminohexane -conjugated HA ledto creating a biphasic stable construct without the need for chemical cross-linker and physical stimuli. The designed scaffolds were tested in the knee joint of rabbits and showed remarkable osteochondral regeneration after 8 weeks.

8. Future direction and conclusion

Bone regeneration is a very complex process that includes various factors, cells, and biomolecules. In large bone defects, the body does not have the ability to heal these injuries by itself. As a result, it needs external interventions. Although extensive methods, including surgical methods and physical methods using different materials, are used to treat bone injuries today, these methods face limitations. The spread of bone diseases with the increase in the elderly population leads researchers to create alternative treatment methods for bone healing. Recently, 3D bio-printing has been proposed for the treatment of large bone injuries, and extensive research has been done in this field in the last few years. This study shows the potential of 3D bio-printing strategies in the creation of different scaffolds for large bone tissue defect regeneration. The use of printed scaffolds containing cells with the ability to imitate natural tissue has opened a window of hope for creating new bone substitutes. Innovation in the use of these methods, such as in-situ printing, pre-vascularization, or the use of multi-matrix scaffolds for the combined treatment of bone and cartilage increases the capability of these structures. There is still a need to investigate and create new scaffolds in this field, which can increase the therapeutic potential of these scaffolds in the future. It seems that 3D bio-printing can provide effective treatment methods for repairing large bone tissues in the near future.