Properties of Scaffold Reinforcement for Tendon Tissue Engineering in vitro Degradation

GONG Ze(龚 泽)，ZHANG Pei-hua(张佩华)* ，WANG Wen-zu(王文祖)，LUO Yun-wei(罗云蔚)

1 Key Laboratory of Textile Science and Technology，Ministry of Education，Donghua University，Shanghai 201620，China

2 Colleges of Textiles，Donghua University，Shanghai 201620，China

Introduction

The tendon defect is one of the common clinical diseases.At present，the most widely used tendons at home and abroad are those artificial ones improved by tissue engineering technology［1-2］.The three elements of tissue engineering include:cell，scaffold，and growth factor［3］.Scaffold，as one of the three elements，must has the following characteristics［4］:(1)good biocompatibility，non-toxic;(2)appropriate threedimensional shape;(3)large pore with good connectivity between them and suitable pore size;(4)appropriate surface morphology and chemical properties; (5) correct fiber orientation and large specific surface area;(6)degradation of the scaffold should be suitable for the formation of the organization，in order to match mechanical properties of the scaffold and the growing tissue.

Artificial tendon scaffold for tissue engineering not only plays a supportive role in maintaining the shape of the original organization，but also plays a template role which provides places for cell boarding， growth， differentiation， and proliferation，in order to guide the regeneration of damaged tissue and control structure of the regenerated tissue［5-6］.Geometrical properties including scaffold porosity，aperture size and thickness are important factors which will affect the degradation performance of scaffold［7］.It is still controversial on appropriate porosity and aperture size for tissue engineering tendon scaffold reinforcement (hereinafter referred scaffold reinforcements).

Polyglycolic acid (PGA)and polylactic acid (PLA)are both typical synthesis and biodegradable materials.With hydrolytic degradation in vivo，the produced intermediate metabolites glycolic acid and lactic acid were expelled after in vivo metabolism［8］.

PGA is a biodegradable synthetic material with good biocompatibility， innocuity， high strength， and high degradation speed.Compared with PGA，PLA has lower strength and slower degradation，and its complete degradation will take 1-2 years［9］.Therefore，PGA and PLA multifilaments were mixed to improve degradation of scaffold reinforcement.Wu et al.［10］studied degradation of braided yarns made of 6-PGA/PLA multifilament in different proportions，but they had not yet carried out in-depth research in degradation of scaffold reinforcement obtained by different braiding proportion.

In this article，braided yarns were made with four different proportions of PGA and PLA multifilament.Then knitting weft plain stitch scaffold reinforcements were braided in smalldiameter circular knitting machines.Degradation variations of mass loss， tensile properties， grams per square meter，thickness，caliber，and porosity of scaffold reinforcements with different braiding proportions were investigated.

1 Materials and Methods

1.1 Materials and preparation

Multifilaments of PGA(8.78 tex/24F)and PLA(6.17 tex/16F)were prepared by Shanghai Tianqing Biomaterial Co.，Ltd.Their tensile strengths were 3.33 N and 2.30 N separately.

(1)The braided yarns were braided in 3，4，and 5 spindles of vertical braiding machines with different proportions of PGA/PLA multifilaments(20 ℃，65% RH).Four kinds of braided yarns as 2PGA/1PLA，2PGA/2PLA，3PGA/1PLA，and 3PGA/2PLA were prepared.The fundamental properties were shown in Table 1.

Table 1 The fundamental properties of braided yarns

(2)The tendon scaffold reinforcements were knitted in a 14 needle small circular knitting machine in 20 ℃，65% RH conditions.

1.2 In vitro degradation

In vitro degradation was simulated by immersing the samples in a phosphate buffer saline (PBS)solution (pH =7.4)and placing them into a carbon dioxide cell culture oven(HH.CP-T 01A，Shanghai Yiheng Science and Technology Co.，Ltd.)with constant 37 ℃.The buffer solution was changed once a week.

1.2.1 Mass loss

Mass loss was evaluated using an analytical balance with an accuracy of 0.001 mg.The sample was vacuum dried in a frozen dryer (FD-10-50， Beijing Boyikang Experiment Instruments Co.，Ltd.)at approximately -50 ℃in a vacuum of 8 Pa and was weighed until weight stabilized and all water was evaporated.The mass loss was determined at different aging time intervals.The overall percentage of mass loss was calculated as Eq.(1):

where M1was initial mass，M2was mass after degradation，and m was mass loss.

1.2.2 Tensile strength

Tensile strength was determined at different aging time intervals.The tensile strength remaining was calculated using Eq.(2):

where F1was initial tensile strength and F2was tensile strength after degradation.

1.2.3 Caliber

Caliber was evaluated using a dividing ruler with an accuracy of 0.1 mm.The sample was flattened to measure its width.The caliber was calculated using Eq.(3):

where W was width of scaffold reinforcement after flatten.

1.2.4 Grams per square meter and thickness

The grams per square meter were weighed.The thickness was measured with thickness gauge directly (CH-10-AT，Shanghai Liuling Instrument Plant).The grams per square meter (G)were calculated using Eq.(4):

where M was mass，L and W were length and width of scaffold reinforcement respectively.

1.2.5 Porosity

The porosity of scaffold reinforcement was calculated by image processing techniques.

1.2.6 Surface structure

The surface structure was observed by optical microscope(PXS8-T).

2 Results and Discussion

2.1 Scaffold reinforcement properties

The fundamental properties of four kinds of scaffold reinforcements were listed in Table 2.As yarn count increased，the caliber，grams per square meter，thickness，and tensile strength of scaffold reinforcements increased obviously，but the porosity decreased.The optical microscope photographs of four kinds of scaffold reinforcements were shown in Fig.1.

Table 2 The fundamental properties of four different scaffold reinforcements

Fig.1 Optical microscope photographs of four kinds of scaffold reinforcements(×10)

2.2 Mass loss

As shown in Fig.2，in the first 3-week，the mass losses were very small.It increased rapidly after three weeks，and it became slow after the 6-week.Particularly，the mass loss of 3PGA/1PLA scaffold reinforcement was the highest，about 80%，and the 2PGA/2PLA scaffold reinforcement was the lowest，about 52%.As the PGA multifilament's degradation speed was significantly faster than PLA multifilament，with the highest PGA percentage composition，the 3PGA/1PLA scaffold reinforcement showed the fastest degradation speed.Besides，it was also known that the 2PGA/1PLA scaffold reinforcement's degradation speed was always faster than that of 3PGA/2PLA.Although their PGA multifilament percentage composition was similar，the braided yarns' count of 2PGA/1PLA scaffold reinforcement was smaller than that for 3PGA/2PLA.

Fig.2 Mass losses of four samples during degradation in pH=7.4 PBS for different periods

2.3 Tensile strength

As depicted in Fig.3，the tensile strength of four kinds of scaffold reinforcements fell sharply in the first two weeks in degradation.The tensile strength of 3PGA/1PLA scaffold reinforcement almost all lost in the 6-week.In the 8-week，the tensile strength remaining of 2PGA/1PLA，2PGA/2PLA，and 3PGA/2PLA were 1.57%，6.78%，and 13.23% respectively.

Fig.3 Curves of tensile strength remaining of four samples

2.4 Caliber

In Fig.4，at the initial stage，the caliber of four samples was diminishing.But it increased rapidly after three weeks.Maybe it has a critical point around the 3-week.From test，we knew the caliber of 3PGA/2PLA scaffold reinforcement could reach 8.3 mm，while the others were around 6.5 mm in the 8-week.

Fig.4 Caliber changes of four samples during degradation in vitro

2.5 Grams per square meter and thickness

Fig.5 Grams per square meter changes of four samples during degradation in vitro

Fig.6 Thickness changes of four samples during degradation in vitro

As depicted in Figs.5 -6，the curves of grams per square meter and thickness of four samples were similar during degradation.In the first three weeks，the grams per square meter and thickness increased slightly，which may be caused by the small mass loss.However，the grams per square meter and thickness decreased more quickly.They decreased rapidly between the 3-week and the 6-week and changed little.After the 6-week，their changes became slow.

2.6 Porosity

In Table 3，at the initial stage，the porosity of scaffold reinforcements decreased obviously as the count increased.Moreover，the porosity decreased in the first three weeks and then increased from the 3-week to the 6-week.After the 6-week，it increased slowly.From Table 3，we know the porosity can reach 95.60%-97.05% in the 8-week.

Table 3 Porosity changes of four samples during degradation

2.7 Surface structure

As depicted in Figs.7 -10，the surface structures of four kinds of scaffold reinforcements changed obviously.From the 0-week to the 3-week，the width narrowed down and the surface became smooth.However，some hairiness appeared on the surface of 3PGA/1PLA scaffold reinforcement.In the 6-week，the pore became larger and the hairiness grew more.In the 8-week，the pore grew farther，but the hairiness reduced.

Fig.7 Surface changes of 2PGA/1PLA (×15)

Fig.8 Surface changes of 2PGA/2PLA (×15)

Fig.9 Surface changes of 3PGA/1PLA (×15)

Fig.10 Surface changes of 3PGA/2PLA (×15)

3 Conclusions

From the 8 weeks in vitro degradation，the first three weeks，the tensile strength decreased rapidly.From the 3-week to the 6-week，grams per square meter and thickness decreased rapidly，but the mass loss，caliber，and porosity increased obviously.After six weeks，changes of all performance tended to be slow.The percentage of PGA multifilament and braided yarn count affected the degradation properties of scaffold reinforcement.

［1］Davis P A，Huang S J.A Biodegradable Composite Artificial Tendon［J］.Journal of Materials Science:Materials in Medicine，1992，3(5):359-364.

［2］Edwards S L，Mitchell W，Matthews，et al.Design of Nonwoven Scaffold Structures for Tissue Engineering of the Anterior Cruciate Ligament［J］.AUTEX Research Journal，2004，4(2):86-94.

［3］Tovar N，Murthy N S，Kohn J，et al.ACL Reconstruction Using a Novel Hybrid Scaffold Composed of Polyarylate Fibers and Collagen Fibers［J］.Journal of Biomedical Materials Research Part A，2012，100(11):2913-2920.

［4］Mathew A P，Oksman K，Pierron D，et al.Fibrous Cellulose Nanocomposite Scaffolds Prepared by Partial Dissolution for Potential Use as Ligament or Tendon Substitutes ［J］.Carbohydrate Polymers，2012，87(3):2291-2298.

［5］Freed C E， Vunjak-Novakovic G， Biron R J， et al.Biodegradable Polymer Scaffolds for Tissue Engineering ［J］.Biotechnology，1994，12(7):689-693.

［6］Mikos A G，Georgios S，Leite S M，et al.Laminated Three-Dimensional Biodegradable Foams for Use in Tissue Engineering［J］.Biomaterials，1993，14(5):323-330.

［7］Mansur A A P，Mansur H S.Morphological Characterization of 3D Porous Scaffold Based on Portland Cement ［J］.Key Engineering Materials，2009，396/397/398:687-690.

［8］You Y，Lee S W，Youk J H，et al.In vitro Degradation Behavior of Non-porous Ultra-Fine Poly(glycolic acid)/Poly(Llactic acid)Fibers and Porous Ultra-Fine Poly(glycolic acid)Fibers［J］.Polymer Degradation and Stability，2005，90(3):441-448.

［9］You Y，Min B M，Lee T S，et al.In vitro Degradation Behavior of Eletrospun Polyglycolide，Polylactide，and Poly(lactide-coglycolide)［J］.Journal of Applied Polymer Science，2005，95(2):193-200.

［10］Wu S Q，Zhang P H，Guo Z.The Properties of Braided Yarns with Different PGA/PLA Proportions in vitro Degradation［J］.Journal of Donghua University:Natural Science，2009，35(3):274-278.(in Chinese)