引用本文： 羅永祥, AshwiniRahulAkkineni, AnjaLode, MichaelGelinsky. 3-D打印：一種個性化制備復雜支架和組織工程植入物的多功能快速成型技術. 中國修復重建外科雜志, 2014, 28(3): 279-285. doi: 10.7507/1002-1892.20140064 復制
Many different rapid prototyping methods,also referred to as Additive Manufacturing or Solid Freeform Fabrication,have either been adapted to or were specifically developed for biomedical applications in the last two decades. The developments,progress in the field and limitations of the different technologies have been described in a variety of publications and reviews over the last couple of years[1-5]. One rapid prototyping method is the so-called three-dimensional (3-D) plotting which is defined as the manufacturing of 3-D scaffolds by dispensing pasty (bio) materials at ambient or physiological temperature. It has some similarities with other extrusion-based methods like the widely used Fused Deposition Modeling (FDM) in which thermoplastic polymer melts are deposited in a comparable manner as strands layer-by-layer to create 3-D scaffolds[6]. The most significant advantages of 3-D plotting over most other rapid prototyping methods is the flexibility concerning the materials which can be processed and the mild conditions which in many cases allows the simultaneous processing of biomaterial pastes and sensitive biological components. This broad applicability will be demonstrated below.
Unfortunately,the rapid prototyping technology is still not clearly defined and therefore 3-D plotting (in the biomedical field) is often also called 3-D printing,direct printing,dispense plotting,or even bioprinting if cells or other biological components are involved.
1 Plotting of calcium phosphates (CaP) and other mineral phases
CaP and other minerals/ceramics as the most important bone filling materials and scaffolds have been extensively studied and used for repair and regeneration of bone defects. Lot of work has also been invested in the fabrication of CaP scaffolds by rapid prototyping including 3-D powder printing and extrusion-based techniques. For the powder printing process,CaP particles are mostly bonded using a liquid binder that is delivered by an inkjet printing head[7]. After that,the binder-free powder is removed and the remaining structure is stabilized,in most cases by sintering,which results in a ceramic body possessing high crystallinity and mechanical stability[8-9]. One of the main disadvantages of this method is the difficulty concerning removing the unbound powder,especially from small pore structures[10]. For the extrusion approach,ceramic inks,highly concentrated,water-based suspensions of β-tricalcium phosphate or hydroxyapatite powder,which are dispensed through a moving deposition nozzle in an oil bath to build a ceramic scaffold[11-13]. After that,the scaffolds were sintered to achieve a ceramic body with high mechanical stability. This process,introduced in the literature also as “robocasting” or “direct write assembly”,is very similar to the technique of 3-D plotting. However,both of the mentioned methods require sintering to obtain certain mechanical strength,which hinders loading of drugs,growth factors,and living cells in the CaP pastes for fabricating growth factor-and cell-loaded scaffolds. In addition,such thermal post-processing prevents the combination of CaP phases with other biomaterials like biopolymer hydrogels or polymer-based composites within one scaffold. Therefore,our group has developed an alternative fabrication method for CaP scaffold by 3-D plotting using an optimized calcium phosphate cement (CPC) paste[14]. The whole plotting and post-processing steps are conducted under mild conditions without any heat treatment,change of pH value,or involvement of organic solvents.
Figure 1 illustrates the process of CPC scaffold fabrication by 3-D plotting. A ready-to-use CPC paste (P-CPC),which consists of an α-tricalcium phosphate-based CaP powder mixture and short-chain triglycerides[15],is filled in a cartridge which is loaded in the plotting unit. By using compressed air,P-CPC strands are extruded and laid down to build a CPC scaffold with pre-designed structure (Fig. 1 a,b). For our work we have used a 3-D plotter (“BioScaffolder”) developed by the German company GeSiM[16]. After plotting,the CPC scaffolds are transferred into water or buffered aqueous solutions for setting and hardening (Fig. 1 c). The final product of the setting reaction of this P-CPC is calcium-deficient hydroxyapatite[15]. After drying at room temperature,the CPC scaffolds are hardened and able to be hand-treated (Fig. 1 d,e). Alternatively,the process can be completely performed under sterile conditions-with setting in cell culture medium at 37℃ and without drying in case of incorporation of living cells or with hardening not in aqueous solution but in a humidified atmosphere in case of loading with growth factors or drugs. Plotted CPC scaffolds with different pore sizes and morphologies can easily be achieved[14] (Fig. 1 f).
Figure1
The process of CPC scaffold fabrication by 3-D plotting. The P-CPC paste used for plotting was filled in a plotting cartridge and tested for its printability ? Uniform CPC strands can be extruded through a stainless steel needle ? A 3-D porous scaffold with pre-designed structure is fabricated by 3-D plotting ? After completion of plotting,the scaffold is transferred into water for cement setting and hardening ? Afterwards,the scaffold is dried at room temperature ? The scaffold is mechanically stable and can be hand-treated ? Scaffolds with different pore size and morphologies can easily be produced ? ?Silicon-based mesoporous bioactive glass (MBG) has a highly ordered mesopore channel structure and hence a very high surface area and pore volume,which makes it having superior bioactivity as well as excellent drug delivery ability[17-18]. MBG have been fabricated as 3-D scaffolds for hard tissue engineering by polyurethane templating. However,MBG scaffolds prepared by this method have weak mechanical strength and suffer from severe brittleness because the sintering temperature is not allowed to be high (normally not higher than 700 ) in case of such mesoporous glasses. In addition,Yun et al.[19] and García et al.[20] prepared 3-D MBG scaffolds with hierarchical porosity using a combination of sol-gel,double polymer templating,and rapid prototyping techniques. Through this method,the obtained MBG scaffold had designed structure and uniform pores,but they were still brittle,not easy to handle,and the mechanical strength was quite low. Herein,our group fabricated MBG scaffolds by 3-D plotting using 14% crosslinked polyvinyl alcohol (PVA) as binder. Through our method,pore size (from 100 micrometer to millimeters) as well as the morphology (from cubic to hexahedral) of the fabricated MBG scaffolds could be controlled over a wide range (Fig. 2). Scanning electron microscopy (SEM) image taken from the surface of MBG scaffolds revealed that the MBG particles were bound together by PVA and formed a very dense and rough surface. In addition,transmission electron microscopy (TEM) image showed the regular mesopores with pore size of 5 nm in the MBG powder[21]. Therefore,our plotted MBG scaffold possessed a hierarchical structure: through regular nano (meso) to controlled macro pores. More important,one of the main advantages of our plotted MBG scaffolds is their high mechanical strength and improved toughness. The compressive strength and modulus of the MBG scaffolds with a porosity of 60.4% are (16.10 ± 1.53) MPa and (155.13 ± 14.89) MPa,respectively. Furthermore,the scaffolds still maintained their bulk morphology after 35% deformation instead of being crushed into powder. Main contribution is the presentence of PVA fibers,which served as binder after heat-induced crosslinking which binds MBG particles into a denser structure[21].
2 Plotting of biopolymers and composites
Whereas in most cases 3-D powder printing is applied for rapid prototyping of ceramic scaffolds,extrusion-based methods like 3-D plotting are common for the fabrication of biopolymer hydrogel constructs. Beside others,alginate has been extensively used for the manufacturing of 3-D biopolymer scaffolds and tissue engineering constructs[22]. Alginate is a linear,anionic polysaccharide. It is a biocompatible and biodegradable material,which easily can form a hydrogel in the presence of divalent cations (such as calcium) at mild pH and temperature conditions. Most groups prepare alginate scaffolds from low concentrated alginate sols (0.5 wt%-4 wt%). When applied in bioplotting,low concentrated alginate sols tend to diffuse after extrusion through the needle and the generated soft strands are not able to support the whole 3-D structure of the scaffold during plotting. Therefore,such alginate sols have to be plotted into calcium ions containing solutions to keep the regular shape of the strands[23],but this bears the risk of blockage of the plotting needle and weak binding between alginate strands of adjacent layers. Herein,in our work,concentrated alginate pastes with alginate concentration of 16.7 wt% were developed for 3-D plotting[24]. The prepared concentrated alginate/PVA pastes were homogeneous and suitable for extrusion even through fine needles leading to stable strands. The plotted scaffold was able to keep the regular shape of the strands and uniform structure of 3-D scaffold while plotting in air (Fig. 3). After the whole scaffold was fabricated,it had to be transferred to calcium ions containing solution for crosslinking of alginate. This method is leading to a homogenous crosslinking and a strong bonding of the different layers. SEM analysis revealed that the microstructure of such plotted concentrated alginate/PVA scaffold is porous because the PVA is dissolved during the alginate crosslinking (gelation) process in aqueous solutions.
Figure3
The alginate/PVA (3 ∶ 1) scaffold observation ? Photograph of alginate/PVA (3 ∶1) scaffold in wet state (before crosslinking and drying) ? Light microscopical image (× 3) of alginate/PVA (3∶1) scaffold in wet state (before crosslinking and drying) ? SEM image showing the surface of plotted scaffold after crosslinking and drying (× 2 500) ? SEM image showing the cross-sections of plotted scaffold after crosslinking and drying (× 2 000) ? ?By physical mixing of MBG powder and the above mentioned alginate/PVA paste composite scaffolds could be achieved by means of 3-D plotting. The MBG powder is entrapped in the calcium alginate hydrogel after gelation and leads to a significantly enhanced stiffness compared to that of a pure biopolymer scaffold[24]. Other groups have combined bioglass with thermoplastic polymers like polycaprolactone and fabricated porous 3-D scaffolds by means of FDM[25].
Gelatin is a natural material derived from collagen,and it contains some of the biological signals of the latter such as the RGD sequence which can promote cell adhesion,proliferation and differentiation[26-27]. Therefore,gelatin has often been utilized for scaffold manufacturing by several conventional methods,e. g. for cartilage[28] and bone tissue engineering applications. However,pure gelatin is not suitable for plotting. In our group,we have developed an alginate/gelatin composite,which is suitable for 3-D plotting because alginate increased the viscosity. The viscosity of Gelatin is temperature dependent. Therefore,for plotting a homogeneous gelatin/alginate blend scaffold,a temperature higher than 37℃ is required. SEM images indicated that the plotted alginate/gelatin blend scaffold possessed open and regular pores,and that the surface of the scaffold was very smooth and dense (Fig. 4). hBMSCs attached and spread well on the plotted gelatin/alginate blend 3-D scaffolds. After a cultivation period of 21 days,cells have proliferated greatly and covered the whole surface and even most of the pores of the gelatin/alginate blend scaffolds (manuscript in preparation).
3 Manufacturing of complex scaffolds and tissue engineering constructs
Whereas many rapid prototyping technologies and the respective instruments have been optimized for manufacturing of only one type of material (e. g. many 3-D powder printers only for processing of ceramic powders),3-D plotting opens up the possibility to create scaffolds from more or less any (pasty) material. Therefore it is easier than with other rapid prototyping techniques to fabricate also complex scaffolds which consist of more than one material-and to combine biomaterials belonging to different material classes like ceramics and biopolymers. The only problem one has to solve is that of stabilization of the materials after the plotting process.
As described above,we could demonstrate that plotted CPC structures can be easily solidified by storage under humid conditions or immersion in aqueous solutions-which initiates the setting reaction of the cement precursor phase. Together with the well-known crosslinking of alginate by divalent metal ions it was possible to create 3-D scaffolds,consisting of interpenetrating networks of CPC and alginate strands because the conditions for stabilization of both materials easily could be combined[29]. Such biphasic scaffolds show interesting mechanical properties with an overall increased mechanical strength and reduced brittleness compared to the pure CPC as well as pure alginate scaffolds (Fig. 5). In addition,the two different materials can be combined in a layered fashion to provide constructs which could be of interest for the treatment of osteochondral defects. Due to a only weak interaction between the CPC and alginate strands again an interpenetrating network has been created in the interface region,leading to a firm connection of both materials (Fig. 6)[29].
One of the biggest problems concerning clinical applicability of tissue engineering is the lack of vascularization. Therefore many groups world-wide are searching for new and better solutions to achieve vascular-like structures which can be integrated in scaffolds and tissue engineered constructs before implantation into the defect site. One promising approach is the fabrication of hollow or cell filled tubes and it could be demonstrated that such structures can be fabricated from alginate hydrogels[30-31]. By applying our highly concentrated alginate/PVA blends and self-made core/shell cones we could print 3-D scaffolds from such hollow strands (Fig. 7)[32]. It could be shown that such hollow fibers can be connected to a microfluidic system and that they can be perfused[33]. This opens up the possibility to create pre-vascularized tissue engineering constructs. For the application in bone regeneration,one can think about the combination of (normal,filled) CPC and alginate-based hollow strands in one 3-D scaffold-which in principal is possible with the technique of 3-D plotting.
4 Conclusion
Beside others,3-D plotting has been developed in the last couple of years to one of the promising rapid prototyping technologies concerning fabrication of both 3-D scaffolds with pre-defined inner and outer morphology and tissue engineering constructs. The mild conditions during manufacturing and post-processing allow the integration of sensitive components like growth factors or even living cells and opens up the possibility of combining different materials like calcium phosphate bone cement with biopolymer-based hydrogel pastes in one scaffold. The latter is of interest for the manufacturing of implants for defects at tissue interfaces,consisting e. g. of bone and soft tissue or bone and cartilage. This rapid prototyping technology permits not only the generation of patient-specific implants with respect to their geometry,but also an individual loading with e. g. autologous biological components like bone marrow or blood fractions during the plotting process. 3-D plotting therefore has a good chance to find its place not only in biomaterials research,but also in regenerative medicine and reconstructive surgery.
Many different rapid prototyping methods,also referred to as Additive Manufacturing or Solid Freeform Fabrication,have either been adapted to or were specifically developed for biomedical applications in the last two decades. The developments,progress in the field and limitations of the different technologies have been described in a variety of publications and reviews over the last couple of years[1-5]. One rapid prototyping method is the so-called three-dimensional (3-D) plotting which is defined as the manufacturing of 3-D scaffolds by dispensing pasty (bio) materials at ambient or physiological temperature. It has some similarities with other extrusion-based methods like the widely used Fused Deposition Modeling (FDM) in which thermoplastic polymer melts are deposited in a comparable manner as strands layer-by-layer to create 3-D scaffolds[6]. The most significant advantages of 3-D plotting over most other rapid prototyping methods is the flexibility concerning the materials which can be processed and the mild conditions which in many cases allows the simultaneous processing of biomaterial pastes and sensitive biological components. This broad applicability will be demonstrated below.
Unfortunately,the rapid prototyping technology is still not clearly defined and therefore 3-D plotting (in the biomedical field) is often also called 3-D printing,direct printing,dispense plotting,or even bioprinting if cells or other biological components are involved.
1 Plotting of calcium phosphates (CaP) and other mineral phases
CaP and other minerals/ceramics as the most important bone filling materials and scaffolds have been extensively studied and used for repair and regeneration of bone defects. Lot of work has also been invested in the fabrication of CaP scaffolds by rapid prototyping including 3-D powder printing and extrusion-based techniques. For the powder printing process,CaP particles are mostly bonded using a liquid binder that is delivered by an inkjet printing head[7]. After that,the binder-free powder is removed and the remaining structure is stabilized,in most cases by sintering,which results in a ceramic body possessing high crystallinity and mechanical stability[8-9]. One of the main disadvantages of this method is the difficulty concerning removing the unbound powder,especially from small pore structures[10]. For the extrusion approach,ceramic inks,highly concentrated,water-based suspensions of β-tricalcium phosphate or hydroxyapatite powder,which are dispensed through a moving deposition nozzle in an oil bath to build a ceramic scaffold[11-13]. After that,the scaffolds were sintered to achieve a ceramic body with high mechanical stability. This process,introduced in the literature also as “robocasting” or “direct write assembly”,is very similar to the technique of 3-D plotting. However,both of the mentioned methods require sintering to obtain certain mechanical strength,which hinders loading of drugs,growth factors,and living cells in the CaP pastes for fabricating growth factor-and cell-loaded scaffolds. In addition,such thermal post-processing prevents the combination of CaP phases with other biomaterials like biopolymer hydrogels or polymer-based composites within one scaffold. Therefore,our group has developed an alternative fabrication method for CaP scaffold by 3-D plotting using an optimized calcium phosphate cement (CPC) paste[14]. The whole plotting and post-processing steps are conducted under mild conditions without any heat treatment,change of pH value,or involvement of organic solvents.
Figure 1 illustrates the process of CPC scaffold fabrication by 3-D plotting. A ready-to-use CPC paste (P-CPC),which consists of an α-tricalcium phosphate-based CaP powder mixture and short-chain triglycerides[15],is filled in a cartridge which is loaded in the plotting unit. By using compressed air,P-CPC strands are extruded and laid down to build a CPC scaffold with pre-designed structure (Fig. 1 a,b). For our work we have used a 3-D plotter (“BioScaffolder”) developed by the German company GeSiM[16]. After plotting,the CPC scaffolds are transferred into water or buffered aqueous solutions for setting and hardening (Fig. 1 c). The final product of the setting reaction of this P-CPC is calcium-deficient hydroxyapatite[15]. After drying at room temperature,the CPC scaffolds are hardened and able to be hand-treated (Fig. 1 d,e). Alternatively,the process can be completely performed under sterile conditions-with setting in cell culture medium at 37℃ and without drying in case of incorporation of living cells or with hardening not in aqueous solution but in a humidified atmosphere in case of loading with growth factors or drugs. Plotted CPC scaffolds with different pore sizes and morphologies can easily be achieved[14] (Fig. 1 f).
Figure1
The process of CPC scaffold fabrication by 3-D plotting. The P-CPC paste used for plotting was filled in a plotting cartridge and tested for its printability ? Uniform CPC strands can be extruded through a stainless steel needle ? A 3-D porous scaffold with pre-designed structure is fabricated by 3-D plotting ? After completion of plotting,the scaffold is transferred into water for cement setting and hardening ? Afterwards,the scaffold is dried at room temperature ? The scaffold is mechanically stable and can be hand-treated ? Scaffolds with different pore size and morphologies can easily be produced ? ?Silicon-based mesoporous bioactive glass (MBG) has a highly ordered mesopore channel structure and hence a very high surface area and pore volume,which makes it having superior bioactivity as well as excellent drug delivery ability[17-18]. MBG have been fabricated as 3-D scaffolds for hard tissue engineering by polyurethane templating. However,MBG scaffolds prepared by this method have weak mechanical strength and suffer from severe brittleness because the sintering temperature is not allowed to be high (normally not higher than 700 ) in case of such mesoporous glasses. In addition,Yun et al.[19] and García et al.[20] prepared 3-D MBG scaffolds with hierarchical porosity using a combination of sol-gel,double polymer templating,and rapid prototyping techniques. Through this method,the obtained MBG scaffold had designed structure and uniform pores,but they were still brittle,not easy to handle,and the mechanical strength was quite low. Herein,our group fabricated MBG scaffolds by 3-D plotting using 14% crosslinked polyvinyl alcohol (PVA) as binder. Through our method,pore size (from 100 micrometer to millimeters) as well as the morphology (from cubic to hexahedral) of the fabricated MBG scaffolds could be controlled over a wide range (Fig. 2). Scanning electron microscopy (SEM) image taken from the surface of MBG scaffolds revealed that the MBG particles were bound together by PVA and formed a very dense and rough surface. In addition,transmission electron microscopy (TEM) image showed the regular mesopores with pore size of 5 nm in the MBG powder[21]. Therefore,our plotted MBG scaffold possessed a hierarchical structure: through regular nano (meso) to controlled macro pores. More important,one of the main advantages of our plotted MBG scaffolds is their high mechanical strength and improved toughness. The compressive strength and modulus of the MBG scaffolds with a porosity of 60.4% are (16.10 ± 1.53) MPa and (155.13 ± 14.89) MPa,respectively. Furthermore,the scaffolds still maintained their bulk morphology after 35% deformation instead of being crushed into powder. Main contribution is the presentence of PVA fibers,which served as binder after heat-induced crosslinking which binds MBG particles into a denser structure[21].
2 Plotting of biopolymers and composites
Whereas in most cases 3-D powder printing is applied for rapid prototyping of ceramic scaffolds,extrusion-based methods like 3-D plotting are common for the fabrication of biopolymer hydrogel constructs. Beside others,alginate has been extensively used for the manufacturing of 3-D biopolymer scaffolds and tissue engineering constructs[22]. Alginate is a linear,anionic polysaccharide. It is a biocompatible and biodegradable material,which easily can form a hydrogel in the presence of divalent cations (such as calcium) at mild pH and temperature conditions. Most groups prepare alginate scaffolds from low concentrated alginate sols (0.5 wt%-4 wt%). When applied in bioplotting,low concentrated alginate sols tend to diffuse after extrusion through the needle and the generated soft strands are not able to support the whole 3-D structure of the scaffold during plotting. Therefore,such alginate sols have to be plotted into calcium ions containing solutions to keep the regular shape of the strands[23],but this bears the risk of blockage of the plotting needle and weak binding between alginate strands of adjacent layers. Herein,in our work,concentrated alginate pastes with alginate concentration of 16.7 wt% were developed for 3-D plotting[24]. The prepared concentrated alginate/PVA pastes were homogeneous and suitable for extrusion even through fine needles leading to stable strands. The plotted scaffold was able to keep the regular shape of the strands and uniform structure of 3-D scaffold while plotting in air (Fig. 3). After the whole scaffold was fabricated,it had to be transferred to calcium ions containing solution for crosslinking of alginate. This method is leading to a homogenous crosslinking and a strong bonding of the different layers. SEM analysis revealed that the microstructure of such plotted concentrated alginate/PVA scaffold is porous because the PVA is dissolved during the alginate crosslinking (gelation) process in aqueous solutions.
Figure3
The alginate/PVA (3 ∶ 1) scaffold observation ? Photograph of alginate/PVA (3 ∶1) scaffold in wet state (before crosslinking and drying) ? Light microscopical image (× 3) of alginate/PVA (3∶1) scaffold in wet state (before crosslinking and drying) ? SEM image showing the surface of plotted scaffold after crosslinking and drying (× 2 500) ? SEM image showing the cross-sections of plotted scaffold after crosslinking and drying (× 2 000) ? ?By physical mixing of MBG powder and the above mentioned alginate/PVA paste composite scaffolds could be achieved by means of 3-D plotting. The MBG powder is entrapped in the calcium alginate hydrogel after gelation and leads to a significantly enhanced stiffness compared to that of a pure biopolymer scaffold[24]. Other groups have combined bioglass with thermoplastic polymers like polycaprolactone and fabricated porous 3-D scaffolds by means of FDM[25].
Gelatin is a natural material derived from collagen,and it contains some of the biological signals of the latter such as the RGD sequence which can promote cell adhesion,proliferation and differentiation[26-27]. Therefore,gelatin has often been utilized for scaffold manufacturing by several conventional methods,e. g. for cartilage[28] and bone tissue engineering applications. However,pure gelatin is not suitable for plotting. In our group,we have developed an alginate/gelatin composite,which is suitable for 3-D plotting because alginate increased the viscosity. The viscosity of Gelatin is temperature dependent. Therefore,for plotting a homogeneous gelatin/alginate blend scaffold,a temperature higher than 37℃ is required. SEM images indicated that the plotted alginate/gelatin blend scaffold possessed open and regular pores,and that the surface of the scaffold was very smooth and dense (Fig. 4). hBMSCs attached and spread well on the plotted gelatin/alginate blend 3-D scaffolds. After a cultivation period of 21 days,cells have proliferated greatly and covered the whole surface and even most of the pores of the gelatin/alginate blend scaffolds (manuscript in preparation).
3 Manufacturing of complex scaffolds and tissue engineering constructs
Whereas many rapid prototyping technologies and the respective instruments have been optimized for manufacturing of only one type of material (e. g. many 3-D powder printers only for processing of ceramic powders),3-D plotting opens up the possibility to create scaffolds from more or less any (pasty) material. Therefore it is easier than with other rapid prototyping techniques to fabricate also complex scaffolds which consist of more than one material-and to combine biomaterials belonging to different material classes like ceramics and biopolymers. The only problem one has to solve is that of stabilization of the materials after the plotting process.
As described above,we could demonstrate that plotted CPC structures can be easily solidified by storage under humid conditions or immersion in aqueous solutions-which initiates the setting reaction of the cement precursor phase. Together with the well-known crosslinking of alginate by divalent metal ions it was possible to create 3-D scaffolds,consisting of interpenetrating networks of CPC and alginate strands because the conditions for stabilization of both materials easily could be combined[29]. Such biphasic scaffolds show interesting mechanical properties with an overall increased mechanical strength and reduced brittleness compared to the pure CPC as well as pure alginate scaffolds (Fig. 5). In addition,the two different materials can be combined in a layered fashion to provide constructs which could be of interest for the treatment of osteochondral defects. Due to a only weak interaction between the CPC and alginate strands again an interpenetrating network has been created in the interface region,leading to a firm connection of both materials (Fig. 6)[29].
One of the biggest problems concerning clinical applicability of tissue engineering is the lack of vascularization. Therefore many groups world-wide are searching for new and better solutions to achieve vascular-like structures which can be integrated in scaffolds and tissue engineered constructs before implantation into the defect site. One promising approach is the fabrication of hollow or cell filled tubes and it could be demonstrated that such structures can be fabricated from alginate hydrogels[30-31]. By applying our highly concentrated alginate/PVA blends and self-made core/shell cones we could print 3-D scaffolds from such hollow strands (Fig. 7)[32]. It could be shown that such hollow fibers can be connected to a microfluidic system and that they can be perfused[33]. This opens up the possibility to create pre-vascularized tissue engineering constructs. For the application in bone regeneration,one can think about the combination of (normal,filled) CPC and alginate-based hollow strands in one 3-D scaffold-which in principal is possible with the technique of 3-D plotting.
4 Conclusion
Beside others,3-D plotting has been developed in the last couple of years to one of the promising rapid prototyping technologies concerning fabrication of both 3-D scaffolds with pre-defined inner and outer morphology and tissue engineering constructs. The mild conditions during manufacturing and post-processing allow the integration of sensitive components like growth factors or even living cells and opens up the possibility of combining different materials like calcium phosphate bone cement with biopolymer-based hydrogel pastes in one scaffold. The latter is of interest for the manufacturing of implants for defects at tissue interfaces,consisting e. g. of bone and soft tissue or bone and cartilage. This rapid prototyping technology permits not only the generation of patient-specific implants with respect to their geometry,but also an individual loading with e. g. autologous biological components like bone marrow or blood fractions during the plotting process. 3-D plotting therefore has a good chance to find its place not only in biomaterials research,but also in regenerative medicine and reconstructive surgery.
