Fiber based tissue-engineered scaffold for ligament replacement: design considerations and in vitro evaluation
Authors: James A. Cooper, Helen H. Lu, Frank K. Ko, Joseph W. Freeman, Cato T. Laurencin
Background:
The demand for anterior cruciate ligament (ACL) reconstruction has risen over the past years because ACL is a major joint stabilizer but has poor vascularization and healing capabilities when it is ruptured. Past prostheses have demonstrated good short-term performance, but lack the biomechanical properties of normal ACL to sustain long-term functionalities. This paper proposed a fibrous, biodegradable ACL scaffold constructed with polylactide-co-glycolide (PLGA) 10:90 using a novel 3-D braiding technology. Their method developed a heterogeneous model that mimics the mechanical properties of native ACL and promotes tissue ingrowth. Their main design focus was based on the effects of architecture, porosity, degradability, and cell source on mechanical properties and cell response.
Method:
Fabrication of the ACL scaffold involved the control of architecture, porosity, biodegradability, and cell source. Architectural design is essential for biological response and long-term stability. The scaffolds have attachment sites at both ends and ligament growth site in the middle. Scaffold parameters were controlled using 3-D braiding machines to fabricate circular braids that varied in braiding angles from 26° to 31° and rectangular braids that varied in yarn density of 9, 30, and 60 yarns/bundle. Porosity was characterized using the Micromeritics Autopore III porosimeter and mechanical properties were measured with the Instron Testing System 1331. The mode and median pore size, pore surface area, tensile modulus, tensile strength, and maximum tensile load were all determined as a function of the braiding angle.
Two cell lines were used in this study: primary ACL fibroblasts isolated from New Zealand White rabbits and BALB/C CL7 mouse fibroblasts purchased from American Type Culture Collection. Both of these cell types were cultured in a-MEM with 10% fetal bovine serum, L-glutamine, and 1% antibiotics. Cells were seeded at 400,000 cells/scaffold and cell growth and morphology were examined after 1 an 8 days using the scanning electron microscopy (SEM).
Results:
Table 1. Summary of porosity data for 3-D circular and rectangular braids
Results from porosity measurement showed that increasing the braiding angle increases the pore surface area but decreases porosity and pore diameter. This is because increasing number of pores will yield smaller pore size. In rectangular braids, increasing yarn density increases total surface area because greater spaces are created between larger yarn bundles that are intertwined.
Table 2. Tensile properties of poly-(a-hydroxyester) yarns and scaffolds
The maximum load of the rectangular braids showed significant change with increasing strain rate. The circular braids could withstand tensile load greater than that of normal human physical activity. The stress-and-strain profile was found to be similar to that of normal ACL.
Figure 4-5. Electron micrographs of BALB/C mouse fibroblast and rabbit ACL cells after 1 day in culture
BALB/C fibroblasts spread readily and perpendicularly to the longitudinal axis of fibers on the scaffold after 1 day of growth. The primary rabbit ACL cells spread much slower and attached unidirectionally along the axis of the fibers.
Figure 6-7. Electron micrographs of BALB/C and rabbit ACL cells after 8 days in culture
After 8 days, BALB/C cells showed no response to the geometry of the scaffold and growth of these cells appeared to be random. The primary ACL cells did not cover the entire scaffold but they did grow allow the geometry of the fibers.
The ACL graft is constructed to match the native ACL by mimicking the architecture of the 3-D collagen fiber matrix and demonstrating similar mechanical properties. Because the past models of ACL grafts failed to sustain for long-term purposes, the model constructed in this study was constructed based on interconnected network of porous structures that will allow for better oxygen and nutrient transportation using the braiding technology. The scaffold is very porous and flexible for better cell attachment and proliferation. The scaffold demonstrated biocompatibility by showing growth of both ACL and BALB/C cells. The primary ACL cells overall grew slower than the BALB/C cells, but responded to the scaffold geometry. Thus, in future studies, the primary ACL cell will be used, but at much higher cell seeding density. Also, the incorporation of growth factors and adhesion proteins on the polymer surface will allow for better cell proliferation.
Critique:
Overall, this paper provided a comprehensive background of the ACL grafts that were developed in the past and the issues and failures they have experienced. It described extensively about the mechanical and architectural design and measurement of the scaffold by showing detailed data of the porosity and mechanical properties. However, it failed to address the fabrication of the actual scaffold. For example, it didn’t address what the 3-D braiding technology was anywhere in the paper, nor did it explain how the braiding angles were changed. This makes it difficult for others to try to reconstruct the model. In addition, the paper didn’t explain the effect of linear density or the purpose of measuring the linear density of the different types of scaffold.
Also, I didn’t find the studies with the incorporation of the two cell lines to be very demonstrative of the effectiveness of the scaffold. First, only 2 cells were used in this study and in conclusion, they mentioned that BALB/C cells will not be used for future studies because they didn’t respond to the geometry of the scaffold. Thus, even though they conducted the 1-day and 8-day studies, the information obtained were not useful. Also, they discussed that the previous models were experiencing difficulty with sustaining long-term functionality. However, the studies they performed only tested short-term performance of 1 and 8 days. Thus, in order to better demonstrate the scaffolds’ performance, they should perform 1 and 2-month studies. In addition, they should also test more cell lines to find the most compatible cell line.
6 comments:
I agree with much of Joyce’s critique. It would be valuable to know how the fibers were aligned, what exactly they were made of, and what information led them to choose the spacing, pore size, and braid size they did. It would be interesting to see if it would be possible to decellularize a native ACL and then seed cells upon this. I wonder why they choose to use a synthetic fiber matrix instead of a native one.
Investigation of other ACL cell types would also be enlightening. It is difficult to discern information from the two cell lines given, especially when only one of them is from a comparable xenogenic ACL tissue. Also, 1 and 8 days seems like a very short amount of time to expect cells to fully seed. I would be interested to see results after a few weeks. More thorough investigation of this setup’s bioreactor could help explain some of the rationale behind timing and cell type.
As a whole the concept is fascinating and useful, particularly in the realm of athletics where this problem seems to be increasingly prevalent.
It's nice how they determined some of the mechanical properties in load bearing applications, but I'd like to see how this scaffold fairs in mechanical wear testing--which is probably one of the most important aspects of the ACL. It doesn't mean much if this can withstand loads higher than human activity but won't last longer than a year. Additionally to mechanical wear, I also have to wonder about the immunogenicity that might result from wear debris since they made no mention of how the polymer was processed in this braid.
@ Carlos: in terms of the braid fabrication steps, I was also very confused when I was reading the paper because they didn't really make any mention of that in the paper. However, I did a little more research and found that they actually published a paper prior to this one that discusses their novel braiding technique.
I am also curious why they chose such a short time-frame. 1 and 8 day study seems like you can hardly get any time-related results from the experiment. And when they say that it can withstand tensile strengths greater than normal activity, what does that mean? I'm guessing that people with torn ACLs are typically loading their ACL with high stress/strain. This seems to need further research to show any promise.
I also agree with Joyce's critique. It is difficult to ascertain how this model would work in vivo without experiments with more cell types and for longer periods of time. Also, it seems that the authors have discarded one cell line for experimentation because it did not yield favorable results. Results are results--and though it would be useful to explore other cell lines, it should still be noted that at least one cell line did not grow on the construct. This could be indicative of insufficiency in the design of the scaffold.
@ Eric: You brought up a good point. I think they mentioned that their circularly braided scaffold can withstand higher load to demonstrate that their synthetic scaffold is mechanistically compatible. However, I completely agree with you that they need to perform further research to show that their scaffold is significantly better than others.
@ Mansi: I agree with you that they definitely need to run more cell lines considering that they rendered one of the cell lines insufficient for future studies.
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