1. Using PLLA Scaffolds in the Restoration of Cartilage Tissue
Miles Quaife, Emmanuel Aidan Acosta, Julian Lu ,
December 1, 2014
I. Problem Area/Scope/ Topic Definition
Defects in articular cartilage tissue (ex. torn labrum, slipped disks, osteoarthritis) are are
a significant medical problem, especially among athletes, the elderly, and “blue-collar” workers.
In the USA alone, over six million hospital admissions are due to cartilage disease (Childs et. al.,
2013, p. 2). Additionally, cartilage is difficult to repair, owing to the fact that articular cartilage
regenerates poorly, and that the stratified structure of cartilage tissue makes its restoration
complex (Childs et. al., 2013, p. 2).
Ideally, treating cartilage involves grafting cartilage tissue from the patient or a separate
donor (Childs et. al., 2013, p. 2). However, this method is subject to a few problems, such as lack
of cartilage tissue from donors, incompatible shape of a cartilage graft, and possible negative
reactions at the site where the graft was taken (Childs et. al., 2013, p. 2). Additionally, the
cartilage that is grafted must be from an area that bears the same load as its destination; cartilage
from a low-load bearing area will not be able to support a high load if placed in an area subject to
heavier and stronger loads (Childs et. al., 2013, p. 2).
These issues can be resolved using tissue engineered cartilage grown from mesenchymal
stem cells seeded in a scaffold (Childs et. al., 2013, p. 2). This allows stem cells to be taken from
the patient, eliminating the need to find a donor or graft chondrocytes from a suitable area (Izal

2. et. al., 2012, p. 1738). Likewise, the scaffold can be manipulated so that it is able to fit the
damaged area (Childs et. al., 2013, p. 2). The material used for the scaffold must be able to
minimize any reaction from the target area, maximize cell growth and proliferation, and get
reabsorbed by the body as soon as the tissue matures (Childs et. al., 2013, p. 2). A material that
fits most, if not all of these categories, is PLLA.
II. Background
Poly (L) - Lactic Acid (PLLA) is a polymer used as a scaffolding material for growth of
various tissues, as well as other medical devices such as surgical anchors, screws, plates, pins,
rods, and bandage mesh (Rafael et al, 2010). PLLA is a choice material for creation of cartilage
scaffolds, due to moderate plasticity for loading stability, natural bioavailability as a biochemical
byproduct in the body from anaerobic respiration, and production from many organic carbon
chained substances. These factors constitute a designable structural material dependent on
polymeric composition. PLLA also has controllable rates of degradation dependent on
compositional blend and amount, allowing seeded cells, time to blend and adhere to the existing
damaged tissue. As the cells grow, the PLLA degrades into monomeric form where it is
reabsorbed into cellular respiration, and no harmful amounts of byproducts are created.
III. Current Methods
Currently, tissue engineering and synthesization are limited by the inability to produce a
3 dimensional model of tissues, which directs cellular growth orientation for macro scale tissues
and organs. Many tissues require not only an accumulation of cells, but also geometric stability
to function properly, especially cartilage. Articulate or hyaline cartilage is a thin layer of tissue

3. found in between joints on the ends of bones, which often is damaged through compressional
stress and mechanical fatigue.
Cartilage is composed of collagen fibrils (structural protein), proteoglycans (heavily
modified protein by carbohydrates), and chondrocytes (cells responsible for proper synthesis and
release of these other components). The ability to heal this damage has been limited to a creating
an autologous implantation, by obtaining a patient's own chondrocytes, allowing to them to 2
dimensionally multiply, then releasing the cells into an agarose fluid suspension. This
suspension, being a 3 dimensional environment, produces hyaline cartilage the chondrocytic
differentiation, and when injected into the knee of the patient, fills in lesions, tears, or
fatigued/defected locations after an appropriate bed has been arthroscopically created during
surgery. shown below in Figure 1 (Shuler et al, 2002).
Figure 1: Autologous Chondrocyte Implantation
Limitations of this process are the time of process for each patient, and the inability to
produce an entire tissue or organ. This procedure succeeds when the injectable solution interacts
and bonds in the environment it has been injected in, which is not guaranteed. Patients must have

4. enough existing healthy cartilage, for the solution to interact with and adhere to, as well as not
subject the treated joint to stressors that could eliminate cartilage bonding.
PLLA scaffolding provides a stable and directional structure for chondrocytic cell growth.
Characteristics of a good scaffold include mechanical strength, biocompatibility,
biodegradability, porosity, and minimal toxicity (Chen at al, 2014). The scaffold needs to have
the ability to both support attachment initially, and continually as population count increases due
to differentiation, while maintaining the ability of the extracellular matrix (ECM) to infiltrate,
provide growth factors, and nutrients to the dividing cells. This ECM consists of the
aforementioned components of cartilage, as well as other growth factors.
Cell sources used to engineer cartilage and tissue growth vary between chondrocytes,
stem cells, and gene modified cells (Chen at al, 2014). It has been shown, that among human
chondrocytes, adipose derived stem cells (fatty tissue), and synovium derived stem cells (a small
layer of tissue in the synovial capsule), synovium derived stem cells have proven to be far
superior for the production of cartilage matrix, including type 2 collagen (the basis for articular
cartilage and hyaline cartilage), and have the greatest ability for chondrogenesis, among the other
stem cell sources (Chen et al, 2014).
IV. Review of new research technology/ Recent Developments
A research experiment was performed to determine growth and differentiation capacity of
mesenchymal stromal cells (MSCs), (adult stem cells), in PLLA scaffolds for potential use in
cartilage disease treatment, shown below in Figure 2 and 3 (Macro and Scanning Electron
Microscope (SEM)). These PLLA scaffolds were prepared using a freezing extraction method,
and particle leaching method. The PLLA films were created by granular fusion in an oven at

5. 200 degrees Celsius between two metallic molds covered in Teflon. These MSC’s were
cultured in PLLA thin films and thin porous membranes to analyze adherence and
proliferation/differentiation, after permeability and porosity were determined for the various
scaffold type. These scaffolds were then maintained in chondrogenic differentiation fluidic
media for a duration of 21 days. Immunohistochemistry (antigen detection (protein markers))
was used to determine apoptosis, proliferation, and chondrogenic differentiation after this time
duration, shown below in Figure 4. It was found that MSC’s uniformly adhered to the PLLA
membranes and films, thereby increasing the elastic modulus of the scaffolds, as well as the
ECM content of aggrecan (cartilage-specific proteoglycan core protein, critical in cartilage
structure), as well as collagen type 1 (most abundant form of structural protein) and X,
associated with new tissue formation in articular cartilage) (Izal, et al, 2012).
Figure 2. a.) PLLA Film b.) PLLA Membrane c.) PLLA Scaffold

6. Figure 3. Scanning Electron Microscope of Scaffolds
Figure 3. a.) Masson’s Trichome: Blue represents Collagen or Bone b.) Immunohistochemistry
testing showing green for positive tests for labeled species.
V. Advantages and Disadvantages of Applications

7. One main strength of PLLA scaffolds is their ideal degradation rate. Richardson et. al.
(2006) noted that PLLA scaffolds have the perfect balance of stability and degradation rate,
which means that they stay stable enough to position the engineered cartilage tissue and break
down once the cartilage tissue fully matures (p. 4070). PLLA’s degradation rate in vitro has been
determined to be around 4 weeks (Stölzel, et. al., 2014, par. 2). Lebourg, Antón, and Ribelles
(2008) attribute the degradation rate of PLLA scaffolds to the high number of ester units per
polymer chain (p. 2207). Izal et. al. (2012) also note that PLLA degrades slower than hydrogel
scaffolds, which allows the cells to properly conform to the desired tissue configuration (p.
1738). The degradation rate of PLLA, along with its relative stability, makes it a good
scaffolding material.
Another characteristic of PLLA scaffolds that make them useful in tissue engineering is
their low potential for inflammation. Richardson et. al. (2006) state that PLLA scaffolds cause
less inflammatory response than PGA and PLGA scaffolds, which degrade faster (p. 4070).
However, it is unclear whether the low inflammatory response is due to the slower degradation
rate of PLLA. Nevertheless, the fact that PLLA causes less inflammation than other scaffolding
materials makes it a good candidate for use in tissue engineering, particularly involving cartilage.
PLLA scaffolds also aid in the formation of cartilage tissue by allowing cells, particularly
stem cells differentiating into chondrocytes, to form extracellular matrix, which aids in the
strength and viability of grown cartilage. A study by Stölzel et. al. (2014) shows that PLLA
scaffolds allow differentiating stem cells to form high amounts of Collagen type I, type II, and
type X, which aids in the formation of intercellular matrix. (para. 10). Izal et. al. (2012) note that
the formation of extracellular matrix ensures that the grown cartilage tissue will be

8. morphologically similar to actual cartilage (p. 1747). Figure 1 shows the collagen fibers present
in the samples of Stölzel’s experiment
Collagen fibers in Extracellular Matrix of cells in PLLA scaffolds (Stölzel et. al., 2014)
PLLA scaffolds are not without their weaknesses. One particular aspect that PLLA
Scaffolds can be improved on is their mechanical strength. While their Stiffness (4-7 MPa) and
Comprehensive stress at 10% strain (0.13-0.18 MPa) are sufficient for most tissue engineering
applications (Budyanto, Goh, Ooi, 2009, p.110), PLLA scaffolds are not as tough as other
scaffold types (Lebourg et. al., 2008, p. 2208). To address that issue, Izal et. al. (2012) suggest
mechanical stimulation of the grown cartilage (p. 1748). Likewise, pure PLLA isn’t as porous as
other scaffolding materials like PGA, but it is very porous when combined with PGA(Lebourg
et. al., 2008, p. 2208). Finally, Stölzel et. al. (2014) note that PLLA scaffolds have a potential to
deform, which may be problematic in the field of tissue engineering (para. 7).

9. VI. Connection to course Content
PLLA scaffolds are specifically used to mimic the functions of an extracellular matrix, or
ECM. In order for a scaffold to be accepted by a patient’s body, it must contain the same
chemical and cellular structure as the desired ECM to the organ’s tissue. Grafting is used to
minimize the overall rejection of the cells binding to the PLLA scaffold. This involves the
transplanting tissue from one area of a patient’s body to another. The grafting process can also
be done through the use of donor tissue.
One of the biggest issues to the introduction of PLLA scaffolds to the body involves the
cells ability to stick to the tissue. According to an article published by Elsevier (2004), cell
growth factors within scaffolds are inhibited by lack of adhesion, due to hydrophobic properties
within artificially produced ECM. To alleviate this issue, the inner surface of the PLLA
scaffolds is lined with collagen fibers. This is normally extracted through grafting local collagen
around the specific tissue type.
Taking into account conservation principles, the amount of cells deteriorating within the
scaffold must not surpass the amount reproducing, and same applies vice-versa. Over, or under
reproduction of cells would eventually lead to the organ ceasing to function. Such complications
do not occur since the PLLA within the scaffold deteriorates at the same rate cells form, in order
to fill the gaps within the ECM.
VII. New Technology
Using PLLA Scaffolding only makes up one component involving the generation, or
regeneration, of healthy new tissue. In some circumstances where localized bone tissue cannot
be substituted via grafting, due to a lack of healthy tissue cells, stem-cells can be used. Cells that

10. fill in PLLA scaffolds must be appropriate for the desired tissue. Removal of cells from a
localized healthy area can create the risk of degradation, while removal of degraded cells can
accelerate degradation even further. In this situation, researchers have relied on MSC
(mesenchymal stromal cells) extracted from adult bone marrow.
This particular cell has the potential to differentiate into a wide range of tissue, based on
its’ lineage. For cartilage, the MSC induces the phenotypic expression of the healthy tissue
replication, as the MSC differentiates into a chondrocyte-like phenotype. The effects of MSC
continue to be explored. So far the combinations of cell scaffold compatibility are limited.
A. Appendix: Sources:
1. Auras, R.,et al (2010) Frontmatter, in Poly(Lactic Acid): Synthesis, Structures,
Properties, Processing, and Applications, John Wiley & Sons, Inc., Hoboken, NJ, USA.
2. Budyanto L., Goh Y.Q., & Ooi C.P. (2009). Fabrication of porous poly(L-lactide)
(PLLA) scaffolds for tissue engineering using liquid–liquid phase separation and freeze
extraction. ​Journal of Material Science: Materials in Medicine, 20(1), 105-111. Retrieved from
http://link.springer.com
3. Chen JL. et al., (2014) Extracellular matrix production in vitro in cartilage tissue
engineering. Journ of Trans Med [Internet]. [Cited 2014 14 Nov] 12(88):1-9. BioMed Central.
London (UK): BioMed Central.
4. Childs A. et. al. (2013). Novel biologically-inspired rosette nanotube PLLA
scaffolds for improving human mesenchymal stem cell chondrogenic differentiation. ​Biomedical
Materials, ​8(6), 1-12. Retrieved from ​http://iopscience.iop.org

11. 5. Izal I. et. al. (2012). Culture of human bone marrow-derived mesenchymal stem
cells on of poly(l-lactic acid) scaffolds: potential application for the tissue engineering of
cartilage.​ Knee Surgery, Sports Traumatology, Arthroscopy, 21(8), 1737-1750. Retrieved from
http://link.springer.com
6. Lebourg M., Suay-Anton J., & Gomez-Ribelles J.L. (2008). Porous membranes of
PLLA–PCL blend for tissue engineering applications. ​European Polymer Journal, 44(7),
2207-2218. Retrieved from ​http://www.sciencedirect.com​.
7. Richardson S. M. et. al. (2006). The differentiation of bone marrow mesenchymal
stem cells into chondrocyte-like cells on poly-l-lactic acid (PLLA) scaffolds.​ Biomaterials,
27(22), 4069-4078. Retreived from http://www.sciencedirect.com
8. Shuler, Michael L., et al. Bioprocess Engineering: Basic Concepts. Upper Saddle
River, NJ: Prentice Hall, 2002.
9. Stölzel K. et. al. (2014). Immortalised human mesenchymal stem cells undergo
chondrogenic differentiation in alginate and PGA/PLLA scaffolds.​ Cell and Tissue Bank,
Retrieved from​ ​http://link.springer.com
B. Appendix: Contributions
Emmanuel Aidan Acosta: Performed research on the chosen topic, compiled Reference list,
Introduced topic in paper, Stated advantages and disadvantages of technology.
Miles Quaife: Performed Research, added references, stated background, current state of the art
work/methods, and review of new technology/methods.

12. Julian Lu: Performed connection to course content, and evaluation of new technology.