SN Applied Sciences

, 2:192 | Cite as

On 3D printed scaffolds for orthopedic tissue engineering applications

  • Nishant Ranjan
  • Rupinder SinghEmail author
  • I. P. S. Ahuja
  • Ranvijay Kumar
  • Jatenderpal Singh
  • Anita K. Verma
  • Ankita Leekha
Research Article
Part of the following topical collections:
  1. 3. Engineering (general)


This paper outlines an in vitro evaluation of 3D printed scaffold of polylactic acid (PLA) blended with hydroxyapatite (HAp) and chitosan (CS) for orthopedic tissue engineering applications. In the first stage, selected composition/proportion of PLA–HAp–CS (based on melt flow-ability, mechanical and thermal properties) was 3D printed with fused deposition modeling (FDM) process. The 3D printed scaffolds were used for process capability analysis to ascertain the industrial usability of PLA–HAp–CS composite scaffold for batch production (especially in assembly applications). Further, in the second stage, an in vitro evaluation was performed to investigate the linkages of fibroblast cells for 3D printed scaffold. The results of study outline the rapid increase in growth of fibroblast cells for FDM-printed scaffolds of PLA–HAp–CS thus ensuring its capability of supporting cell adhesion and cell proliferation. Further, the dimensional variations and Shore D hardness of 3D printed scaffolds are under statistically control, with process capability indices (Cp and Cpk ≥ 1).


Scaffolds Tissue engineering 3D printing Composite material HAp PLA 

1 Introduction

PLA is one of the commonly used polymers in field of tissue engineering for fabrication of biomedical scaffolds and implants [1]. Biocompatible and biodegradable grades of PLA are available in crystalline as well as amorphous form depending upon relative levels of optical isomers present in the molecules [2, 3]. The reported literature outlined that for fast growth of bones/tissues, matrix of PLA is usually blended with biocompatible as well as bioactive reinforcements [4]. The HAp- and CS-based ceramic reinforcements have been used extensively as reinforcements for fabrication of biomedical implants/scaffolds [5]. These reinforcements are useful biomaterials with potential orthopedic, dental and tissue engineering applications due to properties like: bioactivity, biocompatibility and osteo-conductivity [6, 7, 8]. As a composite material PLA, HAp and CS have number of applications in bone regeneration [9, 10, 11].

Usually, a biocompatible substance is defined as “inserts/implants” having no reaction with tissue for imprecise periods of time. This may be due to the formation of a tough layer within implants [12]. Polymers are classified based on the presence or absence of degradation phenomena in a biological environment that is dependent on their belongings to the second- or third-generation biomaterials [13, 14, 15, 16].

Non-degradable polymers are the essential materials which are largely applicable for preparations of medicine as fillers materials, orthopedic implants/scaffolds, ocular lens, heart valves, bone cements and vascular grafts for long-term devices [17]. Sometimes degradable polymeric biomaterials are selected for manufacturing therapeutic provisional devices for prostheses, resorbable 3D porous structures as scaffolds for drug release [18, 19, 20]. Tissue engineering takes advantage of 3D structures that provide a pattern for cells to join, reproduce, preserve their differentiated function and organize in order to restore structure and function to damaged tissues [21, 22, 23]. Essentially, scaffolds act as a synthetic analogue of the natural extracellular matrix [15, 24, 25, 26, 27]. Some studies in recent past have reported Hap- and CS-coated PLA nanofibers for bone regeneration [28, 29, 30, 31, 32, 33, 34].

The literature review reveals the studies on fabrication of biomedical scaffolds by using biocompatible and biodegradable polymers with reinforcement of bioactive ceramics [3, 28, 30, 32, 35, 36]. But hitherto little has been reported on fit and tolerance of 3D printed scaffolds especially in assembly applications, their process capability analysis and surface roughness (Ra) profile of fibroblast cells (for in vitro analysis). This paper is an extension of work reported for fabrication of biomedical scaffolds [37, 38]. In this study, the selected composition/proportion of PLA–HAp–CS as 91–8–1 (by weight percentage) was further used for 3D printing of biomedical scaffolds (in the first stage), and its process capability analysis followed by in vitro evaluation (in the second stage) was supported with rendered images of fibroblast cells and their Ra profiles.

2 Materials and methods

During pilot experimentation, effort has been made to select suitable material composition/proportion for preparations of biocompatible and degradable scaffolds. Initially, blends of PLA, HAp and CS (biocompatible and bioactive materials) were prepared in different weight proportions. In next step, melt flow index (MFI) characterization was performed to investigate flow continuity for feedstock filaments preparations (as per ASTM D1238), followed by investigations of statistical process capability analysis, mechanical and thermal properties for selections of best set of input process parameters of twin screw extruder [37, 38]. For preparations of feedstock filaments of 3D printing, HAAKE Mini CTW, (Thermo Fisher, Germany) was used. The previous studies have reported parametric optimizations of twin screw extruder (for establishing composition/proportion of HAp and CS in PLA) followed by parametric optimization of fused deposition modeling (FDM) setup from mechanical properties view point [37, 38]. These recommended settings were further used for printing of benchmark (flexural sample as per ASTM D790-17) in the present case study. It should be noted that for commercial applications, the customized biocompatible scaffolds must be dimensionally accurate with acceptable hardness. So, experimental study was conducted to establish the dimensional features of the scaffold prepared (from commercial 3D printing view point). The standard dimensions for benchmark were: full length (FL) 125 mm, full width (FW) 12.7 mm and thickness (H) 3.2 mm. Figure 1a–c shows pictorial view of commercial FDM printer, 3D printed flexural samples and scanning electron microscopy (SEM) images of 3D printed fractured samples at different magnifications. It has been reported that best settings for printing of flexural samples on FDM are: Layer height is 0.2 mm, and raster angle is 30° (for tensile properties) and 45° (for flexural properties) with infill percentage of sample which was 100% [37, 38]. These settings were further used to ascertain the dimensional variations and surface hardness of scaffolds. Figure 2 shows process flow diagram for investigations of biocompatible/bioactive customized scaffolds (blends of PLA, HAp and CS).
Fig. 1

a Operational view of FDM. b 3D printed flexural samples. c Top and fractured view of flexural sample at different magnifications

Fig. 2

Process flow for 3D printing of scaffolds by using PLA–HAp–CS composite

3 Results and discussion

As per process flow diagram (Fig. 2), Tables 1 and 2 show the standard deviation (σ) for measured dimensions (DJM) and Shore D hardness of flexural samples (ten different samples) at 95% confidence level which were 3D printed at best composition/proportion (of PLA–HAp–CS) and other input parametric settings suggested in previous studies [37, 38].
Table 1

Standard deviation and margin of error for DJM of flexural samples (as per ASTM D790-17 standard)

Nominal dimensions (DJN)

125 (FL)

12.7 (FW)

3.2 (H)

Standard deviation (σ) for measured dimensions (DJM)




Margin of error at confidence level of 95%

126.328 ± 0.528

12.751 ± 0.0365

3.243 ± 0.0154

In the first stage, based on Table 1, international tolerance (IT) grades for different dimensions were calculated (see Table 2). As observed from Table 2, IT grades for DJN was 12.70 mm, and for all samples, IT grades lie in tolerance zone (IT06–IT12) acceptable for assembly purposes. Further based on results given in Table 1, the process capability analysis has been performed to investigate that whether process is statically capable or not for industrial utility. For given DJN, the number of tolerance unit can be calculated as [39]:
Table 2

Standard deviation and margin of error for Shore D hardness of flexural samples

Mean Shore D hardness


Standard deviation (σ) for observed Shore D hardness


Margin of error at confidence level of 95%

65.25 ± 0.879

$$N = \frac{1000({D}_{\mathrm{J}\mathrm{N}}- {D}_{\mathrm{J}\mathrm{M}})}{i}$$

\(i=0.45\sqrt[3]{D}+ 0.001D\) is the tolerance factor and D is the geometric mean of generic data.

In this case study, for FW of flexural sample, DJN = 12.70 mm (see Table 1). Further, the values of D and i can be evaluated as:

D = \(\surd\)(10 × 18) = 13.416 mm [39] and i = 1.082964.

For flexural sample dimension FW by following Eq. (1):
$$N = 120.$$
The evaluation studies were performed by selecting the ten samples of measured values of “FW.” The outcome of the study is considerable for industrial utility if and only if all the measured values with considerable standard deviation exist in between the upper and lower critical limit [40, 41]. The term process capability indices (Cp, Cpk) are used to summarize a system’s ability to meet two-sided specification limits (upper and lower). Table 3 shows output result of process capability wizard software for nominal dimension 12.70 mm.
Table 3

IT grades for DJN of width 12.70 mm and i = 1.082964

S. no



IT grade




IT 12




IT 10




IT 08




IT 11




IT 11




IT 06




IT 11




IT 11




IT 09




IT 09

As per observations of Table 4, statistical process capability analysis calculations have been made for Shore D hardness value of the flexural sample. Table 5 shows output result of process capability for Shore D hardness value for biomedical scaffolds printed by using FDM.
Table 4

Output result of DJN 12.70 mm of biomedical scaffolds







Maximum (in mm)


USL (in mm)


Average (X-Bar) (in mm)


Target (in mm)


Minimum (in mm)


LSL (in mm)


Data Range


Tolerance (in mm)




AD test


Std. deviation (σ)










USL upper specification limit, LSL lower specification limit, AD Anderson–Darling, Pp process performance, Ppk process performance index

Table 5

Output result of Shore D value for biomedical scaffolds











Average (X-Bar)







Data range






AD test


Std. deviation










In order to evaluate the biocompatibility of PLA, PLA–HAp, PLA–HAp–CS, an in vitro evaluation was designed to investigate the growth of fibroblast cells in 3D printed scaffolds (with macro-pores 150–300 µm, interconnected with micro-pores < 50 µm with average 10% porosity as per ASTM B276) in the second stage. For the experimentation purpose, 5 mm × 0.5 mm size of scaffold was primarily sterilized in autoclave environment under 103,421 N mm−2 pressure at 121 °C temperature for 15 min time interval and further, samples were cured at an ultraviolet light for 30 min before start of actual in vitro evaluation. For this study, fibroblasts cells of mouse embryonic (NIH3T3) were exposed in adherent cultures. Cells of 90% confluency were trypsinized and re-suspended in serum-containing medium, and these first entry cells were seeded at a thickness of 1 × 105 cells/well in twenty-four plates for planned examples and were kept in a 5% CO2-humidified 37 °C in CO2 incubator (Thermo, USA). Initially, cells were grown in a standard culture medium, i.e., in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 2 mM l-glutamine and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin). After the cells were allowed to adhere on the scaffolds, the samples were rinsed to remove the unattached cells. The scaffolds with attached cells were incubated for 2 weeks, and the medium was replaced every 3 days. The cells were observed under the inverted microscope (Olympus CKX41). The specimen was transplanted to another well-enhanced with complete media (second entry) and checked for expansion. This was performed to gauge the quantity of follower cells in a culture well to assess the extent of the surface which is secured by cells. Figure 3 shows expansion of NIH3T3 cells in considered samples. The results of this study reveal the growth of fibroblast cells. It has been observed that fibroblast cells were grew rapidly over the scaffolds. This confirms the biocompatibility of the samples in underneath cell bond and cell propagation. Further, photomicrographs of fibroblast cells were processed with image processing software to see the rendered images (for substrate (A) PLA, (B) PLA + HAp, (C) PLA + HAp + CS on NIH-3T3 cell line) and corresponding surface roughness (Ra) value (Fig. 4). As observed from Fig. 4, Ra value was lowest for PLA + HAp + CS on NIH-3T3 cell line; hence, this may be considered as best scaffold material. However, the growth of fibroblast cells was acceptable for other two cases also. These results indicate prepared scaffolds by 3D printing are suitable for in vivo use in tissue engineering applications.
Fig. 3

Estimation of biocompatibility of a PLA, b PLA + HAp, c PLA + HAp + CS on NIH-3T3 cell line

Fig. 4

Rendered images of fibroblast cells and Ra profile a PLA, b PLA + HAp, c PLA + HAp + CS on NIH-3T3 cell line

Further, in order to evaluate the serum stability of PLA, PLA–HAp, PLA–HAp–CS, a serum stability study was conducted to ensure the stability of serum with the fabricated samples. For the experimentation purpose, the 5 mm × 0.5 mm size of scaffold was primarily sterilized in autoclave environment under 103,421 N mm−2 pressure at 121 °C temperature for 15 min time interval and further, samples were cured at an ultraviolet light for 30 min before start of actual experimentation. It should be noted that glass transition temperature of PLA lies in range of 65–70 °C; therefore, while autoclaving at 121 °C temperature, there will be structural changes. But in scaffold application, the actual temperature will not rise above 40 °C. In the present in vitro testing, autoclaving was performed at 121 °C temperature as a standard procedure being followed in laboratory environment. Fetal bovine serum (FBS) was diluted to 10% using autoclaved phosphate-buffered saline (PBS). The weights of the fabricated samples were taken prior to the start of the experiment. The specimens were kept in six-well plate, and 3 ml of 10% FBS was added to each well. The experiment was continued up to 1 month so as to see the changes in the weight of the fabricated samples. Post 1 month of incubation, the experiment was terminated and again the weight of fabricated samples was calculated and percentage changes in weight were measured (see Table 6). As observed from Table 6, percentage reduction in weight in serum stability test is minimum for PLA + HAp + CS-based samples. Hence, these thermoplastic composites are acceptable in tissue engineering applications.
Table 6

Percentage reduction in weight (based upon serum stability test)

Sample type

%Reduction in weight

PLA (virgin)




PLA + HAp + CS


4 Conclusions

Following conclusions have been drawn from the present study of in vitro evaluation of 3D printed scaffolds:
  1. 1.

    The proposed 3D printing route for preparation of functional prototypes is statistically controlled. (As observed from Cp and Cpk, values are ≥ 1.) Further, these functional prototypes are acceptable for assembly applications as the IT grades are ≤ IT12.

  2. 2.

    The PLA–HAp–CS-based 3D printed functional prototypes are acceptable from biocompatibility and bioactivity view point (as observed from Ra profile, serum stability test) and can be gainfully employed for commercial production in tissue engineering applications. Further, it has been ascertained that in place of PLA, PLA–HAp, synergic combination of PLA–HAp–CS is better solution from biocompatibility view point of functional prototypes.




The authors are grateful to SERB under AISTDF Secretariat (File No. IMRC/AISTDF/R&D/P-10/2017, Dated 01-02-2018).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Nature Switzerland AG 2020

Authors and Affiliations

  1. 1.Department of Production EngineeringGuru Nanak Dev Engineering CollegeLudhianaIndia
  2. 2.Department of Mechanical EngineeringPunjabi UniversityPatialaIndia
  3. 3.Department of Defence ProductionDGQA, SQAE(A)JabalpurIndia
  4. 4.Nanobiotech Lab, Kirori Mal CollegeUniversity of DelhiNew DelhiIndia

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