Tissue engineering applied to the retinal prosthesis: Neurotrophin-eluting polymeric hydrogel coatings

Fig. 1. Multi-electrode polyimide array for retinal prosthesis studies. The array has 15 electrodes each 400 mm in diameter arranged in a 3 × 5 matrix.

Summary:

This article discusses the possibility of improving visual prosthetics for those who have lost or severely damaged their visual ability. The current prospective replacement for a damaged retina (where the photoreceptors are located) is using a multi-electrode polyimide array as seen in the photograph above. One of the problems faced by bioengineers is that this array transmits low resolution due to the relatively large electrodes that require a high stimulation threshold. Scientists cannot reduce the size of the electrodes because a larger size is needed to decrease charge density that would otherwise cause damage to the electrodes and the tissue surrounding it. Additionally, implantation of devices often involves scar tissue that increases the physical separation between the electrodes and neurons, thereby creating an even greater threshold. In this study they use a popular neural tissue engineering material, biodegradable drug-releasing hydrogels, to try to improve these electron-tissue concerns. An advantage of using these hydrogels is that they can be engineered to release neurotrophins (i.e. BDNF, or brain derived neurotrophic factor), which can promote neuron survival and neurite extensions, and can effectively bring the neurons closer to the electrodes in the retina.

This experimental study cultured explants of rabbit retinas in neurotrophin-eluting PEGPLA poly(ethyleneglycol)-poly(lactic acid) hydrogel boluses to study the effect of BDNF-releasing polymer boluses on neurite length. These cultures were then exposed to six different medium conditions: 1540LA2, 1540LA4 BDNF-releasing boluses, BDNF+ control, 1540LA2 and 1540LA4 BSA-releasing shams, and negative control receiving no BDNF for 7 days in vitro. The results of this study indicate that there is a significant increase in neurite length after 7 days in samples exposed to BDNF in comparison to the control study. Additionally, it was found that the polymer itself does not increase neurite length. BDNF was not found to make a significant difference in neurite density. After 14 days, neurite length only increased for BDNF+ samples. Possible explanations of these findings include the possibility that: BDNF affects cell adhesion of the explants to the cell culture layer, or a BDNF concentration gradient caused a slow/stop in neurite growth. These significances led the researchers to conclude that PEGPLA hydrogel boluses can promote short-term neurite extension and that PEGPLA + BDNF boluses can promote extension equal to or better than BDNF being directly injected into the culture medium. On the other hand, a decline in BDNF release induces a retraction of growth in neurites.

Why I chose this article:

Most people consider vision as the most valued of their senses (at least I do). Also, there is much medical and technological advancement that has not been touched upon in the study of vision (e.g. a permanent treatment for glaucoma or corrective vision). This study shows one significant method for increasing neurite growth in the retina, which brings us a step closer to developing a better resolution multi-electrode array for use as a retinal prosthetic. It also provides a vision (no pun intended) for other bioengineers to look for other factors besides BDNF to sustainably extend neurite length. In the very distant future, this could eventually even lead to bioengineering a fully-functional prosthetic eye (not just the retina!)… and wouldn’t that be pretty awesome?

Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature-Responsive Culture Surfac

Tatsuya Shimizu, Masayuki Yamato, Yuki Isoi, Takumitsu Akutsu, Takeshi Setomaru, Kazuhiko Abe, Akihiko Kikuchi, Mitsuo Umezu, Teruo Okano

This paper discusses a new method utilized to fabricate pulsatile cardiac grafts that layers cell sheets 3-dimensionally. By applying poly(N-isopropylacrylamide) (PIPAAm), a temperature-responsive polymer, confluent cells will detach as a cell sheet easily by reducing temperature, forgoing any need for enzymatic treatments. The experiment applied this technique on neonatal rat cardiomyocyte sheets in order to construct cardiac grafts. After overlaying 4 layers, the sheets were observed macroscopically to pulse spontaneously. These grafts were then transplanted into subcutaneous tissue of nude rats in vivo, as well as into 3-week-old and 8-week-old rats. The long-term survival of these grafts was confirmed for up to 12 weeks.

Their results showed that when culture temperature was decreased from 37°C to 20°C, the cardiomyocytes detached as a contiguous cell sheet within an hour without any residual cells remaining on the surface. Electrical communication between the layered cell sheets was monitored on TCPS dishes. Beating of the layered cell sheets initially stopped but began to pulsate spontaneously and simultaneously within one week after layering. Surface electrograms were also used to monitor the cardiac grafts after transplantation into the nude rats. However, these showed that the Graft beating rates varied from 13 to 96 bpm and were relatively slow in comparison with host hearts (332±27 bpm, n=5). Transplantation sites were opened 3 weeks after transplantation to examine the functional effectiveness of the grafts. Their analysis showed that the fractional shortening of the grafts increased depending on the number of layers of the cell graft.

BE115's Tissue Engineering Blog: Tissue Engineering of the Meniscus

BE115's Tissue Engineering Blog: Tissue Engineering of the Meniscus

The Effect of Growth Factor Treatment on Mensical Chondrocyte Proliferation and Differentiation on Polyglycolic Acid Scaffolds.

Growth Factors and Fibrochondrocytes in Scaffolds

Comparison of Scaffolds and Culture Conditions for Tissue Engineering of the Knee Mensicus

Oncogenic Alterations in Metabolism

This review article discusses the fundamental changes in cell metabolism between normal cells and cancer cells. Tumor cells have different metabolic requirements than normal cells; for example, tumor cells typically experience hypoxic environments (either because their size has become too large for efficient O2 diffusion or if they are too far away from a blood vessel). As a result, it is shown that many glycolytic pathway proteins are upregulated as a mechanism to deal with these different physiological environments. For example, hypoxia inducible transcription factor (HIF-1) is shown to be directly upregulated in response to hypoxic conditions. This transcription factor controls the expression of various glycolytic pathway proteins including lactate dehydrogenase and phosphofructokinase L that allow the cancer cell to survive in low O2 conditions.

The tumor microenvironment also has other effects on cell metabolism including angiogenic factors and apoptosis. For example, hypoxia and hypoglycemia stimulate VEGF and other factors for angiogenesis.

The effect of tumor microenvironment on cell metabolism is interesting because these conditions can effectively used in potential future cancer therapeutics. For example, as a result of the upregulation of glycolytic pathway proteins, high lactid acid (from the result of glycolysis and anaerobic respiration) levels are typically found around active tumors (The Warburg Effect). This high acidic environment and other upregulated glycolytic pathway proteins can prove to be effective markers for cancer therapeutics. For example, a Natural Killer Cell or macrophage may be engineered to migrate towards high acidic environments (or to recognize certain glycolytic markers) and may thus facilitate in fighting cancer. The upregulation of glycolytic pathway proteins can also serve as a potential target in preventing developing cancer cells from adapting to their tumor microenvironments. For example, if developing cancer cells are unable to upregulate a vital Glut4 transporter for glycolysis, they may not be able to survive or grow for long.

Three-Dimensional Engineered Heart

Summary:

A new technique was developed that allows neonatal rat cardiac myocytes to form spontaneously and coherently beating 3- dimensional engineering heart tissue in vitro. This new culture system utilizes a 3-dimensional collagen matrix to form a spontaneously and coherently beating cardiac myocyte matrix that offers the following advantages:

1-     A 3 dimensional structure rather then monolayers that better resemble an intact heart tissue

2-     Dedifferentiation and overgrowth by non-cardiomyocytes was inherently inhibited

3-     Gave the opportunity to determine contractile force in standard organ baths

4-     Stable cultivation time and measurement of contractile force

5-     Provide simple genetic manipulation in comparison with intact heart tissue

After 26 days of casting, the contractile activity was monitored in standard organ baths or continuously in a CO2 incubator for up to 18 days. Long-term measurement revealed an increase in force between 8-18 days after casting and stable forces thereafter. On the 10^th day the twitch amplitude of electrically paced engineered heart tissue was 0.51 nN at length of maximal force development in addition to a maximally effective calcium concentration.

Overall the engineered heart tissue retained many physiological characteristics of rat cardiac tissue such as:  A positive force-length and a negative force-frequency relation, high sensitivity to calcium  etc. and allowed efficient gene transfer with subsequent force measurements.

 For your interest here is a short video of the latest development of this topic:

http://www.cnn.com/video/#/video/health/2008/01/15/pkg.rat.heart.update.kare?iref=videosearch

 

Topic was chosen because:

The new cell culture model (3-Dimensional) provides a better physiological way to build a beating heart tissue then the monolayer culture model. In addition, it provides the opportunity to study the consequences of the genetic, mechanical and pharmacological manipulation in vitro under controlled condition. The rat cardiac myocytes were chosen because they are a standard model, well characterized and easy to prepare in large quantities. However, this indicates that engineered heart tissue of different shape could potentially be useful as tissue equivalents for in vivo tissue repairs. This could be a huge breakthrough in medicine as this will greatly increase the life expectancy of many individuals that suffer from heart diseases. 

 

 

 

Grafting of bioactivepolymers onto titanium surfaces and human osteoblasts response

Titanium (Ti) and its alloys are exceptional materials for use in fields such as dentistry due to their resistant to corrosion and its biocompatibility. Though the uses of titanium-based surfaces have been shown to have short term success, problems arise in long run use due to weak integration of implant into the bone tissue. In addition, both specific and non-specific binding of cells and proteins in vivo were reported. In this communication, a titanium surface is chemically modified in order to enhance the absorption of specific proteins and cells. The titanium surface was functionalized with anionic groups by grafting bioactive polymers such as sodium sulfonate. Functionalization was achieved in two steps: surface oxidation with hydrogen peroxide followed by radical polymerization by treatment of sodium styrene sulfonate (NaSS). Human osteoblast-like cells (cell line MG63) were tested for cell adhesion on the modified surface and subsequent mineralization.

A series of analytical techniques were used in order to verify the presence of these anionic groups on the titanium surface: ATR/FTIR allowed for the detection of functional groups; XPS was used for analyzing chemical composition; and profilometry characterized the surface composition. In addition, a colorimetric assay was used to quantify the concentration of functional groups present on the surface. Controls for this experiment included a pure and oxidized titanium surface. MG63 cells were allowed to adhere for 30 minutes and were stirred for 15 minutes thereafter to remove weakly adhered cells.

ATR/FTIR and XPS verified the presence of the functional group on the grafted surface. Profilometry showed that the initial oxidation step resulted in a rough surface while the subsequent step lead to a smoother surface. The functional group was highly concentrated on the grafted surface based on the results of colorimetric assay. The graph showed a statistically significant amount of MG63 cells being adhered to the grafted titanium when compared to both the pure titanium and oxidized titanium surfaces (Fig. 4). However, though the mean amount of mineralization is higher in the grafted titanium case when compared to other two controls, it is difficult to conclude whether these results were statistically significant due to the overlapping error bars (Fig. 5).

Synthetic chemistry allows for grafting of bioactive polymers onto metal surfaces, such as titanium, that can introduce a huge array of functionalities. Though there are huge constraints, such as having bioorthogonal functional groups, that limits the general scope of this technique, various modifications can allow for several other applications beside mineralization.

Cell-based tissue engineering for lung regeneration

Emphysema is a lung disease that results in permanently decreased lung volume and tissue loss. The authors of this paper evaluated a potential treatment of emphysema using tissue engineered scaffolds made from Gelfoam sponge. The purpose of the scaffold was to provide a temporary, biodegradable structure that would allow for migration of lung cells and the formation of new vasculature to help regenerate lung tissue. Gelfoam sponge is as a soft and pliable hemostatic material commonly used in surgeries. If the amounts of Gelfoam sponge used is small, it can be completely broken down and absorbed by the body over the course of 2-6 months, usually with minimal immune response and little or no scarring.

Small pieces of the sponge were implanted into the lungs of rats to determine how cells grew into and around the device. In 2-4 weeks, cell migration into the device was observed. After six months, the sponges completely dissolved and some cells remained in the area, but there was no new vasculature and no normal lung structure formed.

The experiment was repeated using sponges that had been seeded with progenitor cells that were isolated from fetal rats. After 100-128 days, the sponge began to degrade, but unlike the unseeded sponges, vascular-like structures were observed where the cell seeded sponge had been.

Fig. 1 Remodeling of sponge and formation of “alveolar-like structures.” A–C, day 100; D–F, day 128. In 100-128 days, the sponge degrades, leaving structured “lung tissue” in its place. F, surrounding alveolar units. Thin arrows indicate the vascular-like structures inside the sponge area, and open arrows indicate epithelial-like cells in sponge.

India blue ink was injected into the pulmonary artery and ink was seen in the sponge, indicating that the structures were connected to the circulatory system. The rats were injected with BrdU 24 hrs before being sacrificed and proliferation and division was observed in the cells within the sponge. An immunostain for proSP-C, a marker for type II epithelial cells, revealed the presence of epithelial cells within the sponge. The cells were also stained with an anti-CCSP antibody. CCSP-expressing cells are known to be important for epithelial cell renewal and these cells were also observed in the sponge. Interestingly, many of the cells that grew and proliferated in the sponge appear to be endogenous cells, not the seeded fetal progenitor cells. The researchers speculate that the fetal cells were releasing growth factors and these soluble factors encouraged other cells to migrate into the sponge.

Fig 2. Angiogenesis and immunohistochemistry staining in the
implanted sponges. A and B: India ink pigment found
inside the sponges (thin arrows). C: BrdU staining demonstrates
cell proliferation (thin arrows). D: anti- Clara cell secretory protein antibody (CCSP) (brown color, thin arrows) to demonstrate bronchiolar epithelial cells. E: alveolar
epithelial cell staining (proSP-C; thin arrows). F: endothelial cells were
stained with antibody for von Willebrand factor (VWF; thin
arrows). G: anti-CD45 antibody was used to illustrate the lack
of infiltrating leukocytes in the sponge. H: negative controls for each
antibody.

I found this paper interesting because it demonstrates that using a combination of biological factors and mechanical engineering can create a more effective treatment. A total artificial lung would be very difficult to create because it would require the formation of a complex, organized 3-D structure that is capable of connecting to both the vascular system and the air exchange structures. Also, since the lungs are repeatedly expanded and contracted, a permanently implanted material would have to be biocompatible, flexible and strong and ideally, remain flexible and strong for the patient’s lifetime. If the growth of new tissue can be encouraged and the breakdown products of the implanted material are biodegradable, this could allow some patients to avoid transplants and immunosuppressive drugs and not worry that their artificial lung might fail.

The potential of amniotic membrane/amnion-derived cells for regeneration of various tissues.

The amniotic membrane and the cells derived from the amnion, due to their similar characteristics with stem cells, have been reported to be a novel source of generating biological substitutes. Although stem cells have been known as a source of cells for regenerative medicine, the adult stem cells are hard to isolate and grow in culture, while the embryonic stem cells risk tumor formation. Amnion-derived cells, on the other hand, have the following advantages. They have low immunogenicity and anti-inflammatory functions; they are non-tumorigenic; and they involve little ethical problems with usage because these membranes are usually discarded after parturition and can therefore be obtained without harming mothers or babies. In addition, amniotic membrane-derived cells possess multipotent differentiation ability. They can differentiate both in vitro and in vivo into chondrocyte-like cells and three germ layers: endoderm for hepatocytes and pancreatic β-cells, mesoderm for cardiomyocyte-like cells, and ectoderm for neural progenitor cells. All of these advantages sound promising.

The clinical application of amniotic membrane includes the filed of ophthalmology and hyper dry amnion. Studies have shown that the derivative of amniotic membrane promotes the proliferation of bovine cornea epithelial cells. The hyper dry amnion is easy for handling, decreases the degradation of tissue-protein, and has produced good results to patients. With the usage of amnion-derived cells, developing therapeutic strategies such as artificial organs, cell transplantation, and gene therapy will only be more achievable.

This paper is a great recap of what we have learned in the course. Not only can we review the methods and techniques we’ve gone through in some of the labs under the “isolation and cultivation of amniotic cells,” but we also see how significant the field of tissue engineering will be. The potentials of amnion-derived cells ensure more possibilities in reconstruction of damaged tissue and will definitely benefit more populations in the near future.

Optimizing Normoxic Conditions in Liver Devices

The authors of this paper experimented with the possibility of improving oxygenation to hepatocytes in a liver replacement device by adjusting the porosity of the extracellular matrix that said hepatocytes were embedded in. The investigators cultured Sprague Dawley rat hepatocytes on ECM gels constituted from collagen gel, with the addition of sterilized hollow polystyrene microspheres in varying degrees with different cellular densities as well as heterogeneous densities . The gels were also constructed in two different forms, a sandwich configuration as well as homogeneous dispersal. Oxygen transport visualization was accomplished with the addition of dichlorotris ruthenium hydrate, as the dye is sensitive to the presence of molecular oxygen. The cultures were then exposed to an atmosphere of 95% oxygen/5% carbon dioxide. Evaluation of cell viability was done through optical examination as well as Flouview and Metamorph Imaging software. Metabolic status was monitored by NADH autofluorescence and ROS generation. Results showed that the highest oxygen transport distances were achieved with the highest concentration of microspheres (40uL/mL) with an accompanying relative higher oxygenation at distances further away from the origin of oxygen. This is regardless of the configuration of the tissue culture. Examination of cell viability also revealed a definable distance limit at which the majority of cells beyond the limit died. The 40uL/mL enhanced ECM resulted in a 200% increase of this boundary in comparison to normal collagen ECM, from 150 um to 420 um or higher. NADH fluorescence analysis again showed that the oxygen concentration as a function of distance from oxygen origin decreased at a much slower rate in the 40uL/mL eECM. ROS generation was measured to decrease just as much as in lower or no enhancement; however, absolute values were measured to be less per cell.

The successful development of a bioartificial liver assistive or replacement device is crucial due to susceptibility of the liver to damage. With its various roles in metabolism, detoxification, circulation, and biochemical regulation, the liver is exposed to almost all harmful substances that can pass through the body. In addition, the spread of viral hepatitis has results in the same magnitude of annual deaths as HIV/AIDS, with a large portion of the infected population unresponsive to current treatments. At the same time, one of the difficulties in keep artificial organ tissue alive is proper oxygenation. Hepatocytes are particularly demanding in terms of oxygenation; the liver as a whole is one of the largest consumers of oxygen in the body. This paper shows that it is possible to modify the hepatic scaffolding in a conceptually simple manner to minimize not only hypoxia but also hyperoxia, which can also cause tissue damage in the form of free radicals. As a result, the lifespan of BLADs can be extended, accompanied by a noticeable increase in hepatocyte metabolic performance. This ECM modification is applicable to improving the engineering of other complex, multi-unit structures such as the kidney.

Inhibition of telomerase activity in malignant glioma cells correlates with their sensitivity to temozolomide

Title:	Inhibition of telomerase activity in malignant glioma cells correlates with their sensitivity to temozolomide.
Source:	The British journal of cancer [0007-0920] Kanzawa, T yr:2003 vol:89 iss:5 pg:922 -9

Temozolomide (TMZ) is a drug that has proven to be effective in treating malignant gliomas. O6-alkylguanine alkyltransferase (AGT), a DNA repair protein, reduces the antitumor efficacy of TMZ. Therefore cells with low levels of AGT are sensitive to TMZ (U373-MG and U87-MG cells), while cells expressing high levels of AGT are more resistant (T98G). Because it has been demonstrated that chemosensitivity of tumor cells is associated with a decline in telomerase activity, this study investigates whether the responsiveness of malignant glioma cells to TMZ is associated with the level of telomerase activity.

This study assays for cell viability using trypan blue dye exclusion assay, for cell cycle DNA content via FACScan, for telomerase activity via PCR amplification of telomerase extensions, for hTR and hTERT RNA expression (components necessary for telomerase activity) using RT-PCR, and for transcriptional activity of the hTERT promoter-luciferase constructs using a Microtiter Plate Luminometer. And results show that in low levels of AGT, TMZ affects cell viability in a time- and dose- dependent manner, that there appears to be a TMZ induced G2/M arrest in cells, and an inhibition of telomerase activity, hTERT mRNA expression, and transcriptional activity of hTERT promoter. In conclusion, treatment with TMZ inhibits telomerase activity as it interferes with binding sites of a transcription factor, Sp1.

The importance of these findings suggest that TMZ therapy could be determine by monitoring telomerase activity. Therefore the efficacy of TMZ treatment can be quantified.

Characterization of paclitaxel sensitivity in Human glioma- and medulloblastoma-derived cell lines

Overview
This paper examines the sensitivity of a Paclitaxel on gliomas. Paclitaxel is a cytotoxic product that disrupts microtubule integrity and is currently being evaluated for its purpose in killing malignant astrocytic gliomas and medulloblastomas. In the past, methylating agents have been used to form alkyl base adducts in the DNA of the gliomas and medulloblastomas, to prevent growth of these tumors, and induce death. However, because of the processes of evolution most malignant tumors have become resistant to this treatment.

Since damaging the DNA no longer has an effect on malignant tumors, the scientists in this paper have used a different drug that targets the cytoskeleton of the cell, rather than it’s DNA. Experiments with Paclitaxel has shown that the drug works on several malignant tumors including those that are resistant to the effects of methylating agents on tumor DNA. By damaging and preventing cytoskeleton growth and rearrangement, Paclitaxel prevents the cells from undergoing all the steps of mitosis, as the cytoskeleton plays a key role in cell replication and growth. When a cell is prevented from entering or completing mitosis, it will eventually enter apoptosis.

Specificities of Experiment:
Medulloblastoma cells and glioma cell lines were taken from several different sources. They were plated individually on 12 well plates at a cell count of 750cells/well and incubated for 4,8,24, and 48 hours. They tested the sensitivity of Paclitaxel by conducting a clonogenic assay.

Results:
An interesting result was that all cell lines displayed biphasic survival curves. The biphasic survival curves are an indication of subpopulations in the cell line that differed in their sensitivity. These biphasic curves were evident throughout most of the beginning time intervals. At later time intervals (24 hrs) the resistant cell lines were eliminated and cell lines showed a single linear survival curve. This result shows that Paclitaxel is effective in eliminating resistant cells. At 48 hours, Paclitaxel did not eliminate any more differences in the sensitivities between the cell lines.

The differences in sensitivity were attributed to not only the biological nature of the specific subpopulations but also the phase of mitosis they were currently in when the drug was added. Those in the G2 phase were more sensitive to Paclitaxel, because of the necessity for microtubules at this stage. However, cells that were in G1 or S phases had a higher resistance to Paclitaxel because a damaged cytoskeleton at this stage in the cycle does not have as catastrophic effects as in other stages.

microRNA's join the p53 network - another piece in the tumour-suppression puzzle

MiRNA’s are small non-coding regulatory RNA’s that mediate post-transcriptional silencing. They show reduced expression in tumor cells and the components of miRNA biogenesis machinery are suppressed in cancer cells. Recently five independent studies have shown that miR-34 (a miRNA ) is a component of the p53 tumor-suppressor network. The p53 network was thought to consist of only proteins and is activated by many cancer associated stress signals like DNA damage, oncogene activation and hypoxia. MiR-34 was identified as a p53 transcriptional target that can regulate cell proliferation and cell death.

Over expression of miR-34 in fibroblasts and tumor cell lines produced cell arrest followed by apoptosis in some of the cell lines. Microarray analysis showed that miR-34 down-regulated hundreds of mRNAs for cell cycle regulators. Minimal miR-34 deletions have been found in human breast and lung cancer cells and alterations in miR-34 are found in tumor cell lines that have wild-type p53.The discovery that miR-34 was part of the p53 network also cleared up a long standing mystery of the speed at which activated p53 was able to repress a large number of genes.

Comparative analysis of genomes shows that non-coding RNA’s are more represented in complex genomes. Non-coding RNA’s are being recognized as key players in tumor development and since they can affect many targets they can possibly regulate many aspects of a particular cellular process simultaneously.

I chose this paper because it gave me a new understanding of a well known tumor suppressor pathway. Even though miRNAs were first discovered about 15 years ago we still do not completely understand their full function. Scientists have identified over 200 different miRNAs with very specific regulatory functions and I hope that some of these will help us understand how to fix cells when they become cancerous. Lin He, one of the authors of this paper just joined the MCB Department at Berkeley.

Magnetofection-- a novel tool for gene transfer

Summary:

Transfer of whole genes rather than protein products has become a useful and valuable technique for gene analysis as well as a promising tool for genetic therapy. Although many viral and non-viral systems for such transfection have been proposed finding an ideal system that can efficiently carry out a gene transfer has been difficult to come across. While viral systems have been found to be highly efficient in transfection, their oncogenic properties, immunogenecity and other such disadvantages limits their use. Non-viral techniques, on the other hand, while low in toxicity and immunogenecity, usually result in inefficient DNA uptake and expression.

In this paper, a series of experiments were conducted to investigate the use of magnetofection using iron oxide (Fe3O4) nanoparticles as a novel non-viral technique for gene transfer of LacZ, and enhanced green fluorescence protein (EGFP) into mice osteoblasts and He99 lung cancer cells respectively.

First, the effect of the nanoparticles on cell viability was tested using diphenyltetrazolium bromide assay and cytotoxicity was not found at any of the Fe3O4 concentrations tested.

Endocytosis of the gene mediated by the nanoparticles was then tested by adding liposome-enveloped LacZ/Fe3O4 to osteoblasts cultures. Half of the cultures were placed above 3599 gauss magnets for five minutes while the other half was not. Successful transfection and Lacz expression was detected after 10 days in both groups (showing that endocytosis has occurred) but the expression was visibly higher in the cells that were exposed to the magnetic field.

Transfection of He99 lung cancer cells with EGFP was then performed. The cells were transfected with one of the following preparations: homemade liposome-enveloped EGFP-DNA with iron oxide nanoparticles, homemade liposome-enveloped EGFP without the nanoparticles, lipofectamine 2000-enveloped EGFP-DNA (positive control), or EGFP-DNA gene alone. Half of the cells were exposed to magnetic field while the other half was not. Expression of EGFP was observed and analyzed. The group found that both commercial EGFP, and homemade liposome-enveloped EGFP/Fe3O4 were successful while the EGFP gene alone and the EGFP without the nanoparticles did not show any transfection. Though transfection occurred both with and without the presence of a magnetic field, like before, the rate was higher when exposed to magnetic field. This was taken to be evidence that due to gravity, just the presence of magnetic nanoparticles results in better transfection.

Why this paper?

Magnetism and nanoparticles are topics that have traditionally fallen in the realm of physics and material science. This paper showcases magnetic nanoparticles under a new light and provides evidence for its effective use in molecular biology and tissue engineering. Nanoparticles are biodegradable, and the technique is very efficient making it ideal for use in tissue engineering, gene analysis, and gene therapy. I found the concept to be very interesting and the same time logical and fairly easy to understand. I expect that in coming years this innovative technique will be perfected and widely used in many fields of molecular biology and genetics.

Targeting Expression with Light Using Caged DNA

Summary:

Gene therapy is one of the most promising prospects in the biomedical and bioorganic realms. However, success within in vivo gene therapy is restricted by two significant limiting factors: site-specific gene delivery and subsequent localized expression within the target cell population. Conventional molecular approaches to manipulating genes in live embryos and animals do not allow for precise up-or-down regulation of gene expression on an individual cellular basis. One novel approach to overcome these obstacles involves the use of caged compounds, which possess a covalently attached group that can be released upon exposure to various wavelengths of light. The bound substance deactivates the primary molecule by inducing unfavorable physical and chemical conformations until photoactivation. Upon release of the photolabile aptamer, the previously inert compound (e.g. oligonucleotides, transcription factors, and DNA/RNA-dependent enzymes) initiates specific cellular pathways for regulated gene expression.

In this report, researchers utilized caged plasmids coding for luciferase and transfected into roughly 1-cm diameter targets in the skin of rats via gold particle bombardment. They hoped to characterize the potential for light-induced expression with higher spatiotemporal control and targeting. The plasmids were rendered inert by 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE), a photoactive caging group. The effectiveness of the DMNPE cage groups is evident with unchanged luciferase expression levels and equivalent to that of nontransfected skin sites. Upon exposure to 355-nm laser light, the uncaged plasmid induces increased luciferase expression within the target sites. Progressively increasing levels of laser dosage generates proportional increases in luciferase production.

Figure 1: Effect of light on luciferase expression in rat skin. Particle-mediated transfection of caged and native pCEP-luciferase was performed on ~1-cm diameter sites in rat skin. Luciferase expression of skin sites transfected with caged plasmid is equal to levels in nontransfected skin. Exposure of skin sites transfected with caged plasmids to increasing amounts of 355-nm laser light increases expression to 6 ± 3, 12 ± 4, and 17 ± 6% of control, respectively. The right bar indicates highest expression (40 ± 12% of control) from skin sites transfected with caged plasmids exposed to light before delivery. The asterisks indicate difference from no light exposure by two-way repeated measures analysis of variance (n = 4; mean ± S.E.).

In addition, DMNPE-caged GFP (green fluorescent protein) plasmids were introduced into HeLa cells through liposome-transfection. Further analysis revealed that the presence of the DMNPE cage groups reversibly blocks gene expression. GFP expression levels remained constant between transfected and normal cells. With application of the 355-nm laser light, GFP expression is subsequently induced within the target HeLa cell populations. Parallel in vitro research confirmed the results from both cases above and suggested regulation of gene expression at the transcription level due to reduced fabrication of mRNA from the GFP plasmid.

Figure 2: Effects of light on native and caged GFP expression in HeLa cultures. HeLa cells were liposome-transfected with caged and native GFP plasmids. The expression level of native pGFP was 43 ± 4.3% of cells (n = 11, mean ± S.E.). Percentages of expression were normalized to this group. Without exposure to light (i.e. with 0 J/cm² of 365-nm light), the fraction of HeLa cells that express caged pGFP (solid bar) is 25.8% of native plasmid expression levels (n = 7, gray bars). After exposure to 0.25 or 0.5 J/cm² of light, expression of the caged material increases to 50% of control. The asterisks indicate significant difference from expression of caged plasmids that received no light exposure (p < style="">t test). Cultures transfected with caged pGFP and treated with 2.8 and 5.6 J/cm² of light showed decreasing expression levels of 20 and 10%, respectively. Native GFP expression levels also decreased with increasing post-transfection, light exposure, from a normalized 100% with no light exposure to 81, 24, and 10% with a light exposure of 0.5, 2.6, or 5.6 J/cm², respectively. Cultures exposed to 2.8 or 5.6 J/cm² of light after transfection showed significantly lower levels of expression than those that received no light (denoted by crosses, p < style="">t test). Plasmids exposed to the highest dose of light (5.6 J/cm²) before transfection (bar labeled Pre-Flash) express at levels equal to control plasmids that received no light.

Why I Chose This Paper:

Light regulated gene expression has the potential to offer unprecedented spatiotemporal control in a wide variety of applications including genetics and cancer research. Different spectra of light can penetrate the skin of the human body without toxic side effects to the surrounding tissue. As the database for available photoactive chemical aptamers and corresponding carriers becomes more firmly established, scientists may finally gain the ability to control differentiation within targeted cells (i.e. stem cells) with exact timing and activation of the correct biochemical pathways. Each year, more than 1 million American citizens are diagnosed with some form of cancer. The National Cancer Institute estimates that nearly 10.5 million people are now living with a previous diagnosis of cancer. Current technology and techniques are limited by the cancer type, location, and progression of the tumor. Devastating side effects along with unsatisfactory success rates have forced scientists into searching for remedies with minimal side effects and overcomes non-specific targeting of healthy cells. Existing technologies and treatments (i.e. radiotherapy, chemotherapy, and surgery) are often administered in combination to increase overall success rates. Possible advancements in cancer research involve nanoparticle carriers with payloads of modified oligonucleotides (i.e. siRNA) or powerful anticancer drugs attached by photoreactive linkers. Photonic excitation and ensuing release would then occur in localized regions of the cancerous tissue with minimum damage.

Stem cell-based tissue engineering with silk biomaterials

Silk is an excellent material to work with in tissue engineering as it is biocompatible, lightweight, strong, elastic, and even thermally stable up to 250°C. Biomaterial scaffolds form an important structural basis for the in vitro and in vivo growth of a variety of engineered tissues. They must be biocompatible, biodegradable, effectively mimic cellular environment (i.e. support cell attachment, migration, etc.), and be versatile in processing options to alter structure and morphology related to tissue-specific needs.

As a 2D film, silk is comparable to collagen films in terms of supporting attachment, spreading and proliferation of fibroblasts. Silk from cocoon fibers was found to support the attachment and growth of human and animal cell lines, due to the positively-charged residues in the fibroin sequence and the negatively charged surface of mammalian cells. The biomedical applications of silk fibroin films could be broadened by surface modifications with RGD or specific growth factors, as well as by blending with other natural or synthetic polymers, like cellulose, collagen, or polyacrylamide.

Hydrogels can be formed from regenerated silk fibroin solution by sol-gel transition in the presence of acid, ions, and other additives. As a hydrogel, silk has applications in guided bone repair, drug release/delivery, and cartilage tissue engineering. In one experiment, silk fibroin hydrogels were used to repair confined, critical-sized cancellous bone defects in a rabbit.

In the form of a non-woven mat or net, silk is incredibly strong and functions well in wound dressing, skin repair, and tissue engineering. It has been shown that non-woven micro-fibrous nets support the adhesion, proliferation, and cell-cell interactions of a wide variety of human cell types including epithelial cells, endothelial cells, glial cells, keratinocytes, osteoblasts, and fibroblasts. Silk fibroin mesh implants were shown to be highly biocompatible and integrated with the surrounding tissue with no apparent degradation. Non-woven nano-fibrous nets and mats are also of interest for biomedical applications because of the material’s high surface area. It supported the attachment, spreading, and proliferation of human bone marrow stromal cells, keratinocytes and fibroblasts.

However, perhaps the most versatile and useful forms silk fibroin is able to take is a 3D porous sponge. Not only is this form able to be molded into endless shapes and structures, it is very strong and contains high porosity and poor interconnectivity. This structure is ideal for bone and cartilage tissue engineering. One study explored the potential of native silk fibroin fibers as 3D scaffolds for tissue engineering of ACL in cultures with dynamic mechanical loading. After weave together these fibers in a manner mimicking the structural assembly of the ALC, MSC were successfully supported in their attachment, spreading, proliferation and differentiation. A great variety of other “yarn” 3D structures have been created with different weaving patterns and subsequent mechanical properties. 3D porous silk fibroin scaffolds have also been used for MSC-based bone tissue engineering; it provided an appropriate environment for MSC’s to proliferate and differentiate and showed promise for repair of critical sized bone defects. 3D porous silk fibroin sponges are also useful in cartilage tissue engineering. Adult articular cartilage has limited self-repair capacity, and previous uses of collagen- and alginate-based scaffolding proved insufficient due to rapid degradation. The useful combination of high strength, porosity, processability, good biocompatibility and ability to support cell adhesion, proliferation, and differentiation suggests 3D porous silk fibroin scaffolds are prime candidates for stem cell- and chondrocyte-based cartilage tissue engineering.

I feel that this paper provides an excellent example of the crucial interplay between materials science and tissue engineering. Adequate scaffolding is becoming a necessity in modern-day tissue engineering, and a variety of materials have been studied and used for supporting cell growth and differentiation. However, none have proven as versatile and innately biocompatible as silk fibroin. The diversity of tissue-modeling possibilities and medical applications allowed by this one material is astounding. I also love how this paper provides an instance of biologists, chemists, and engineers collaborating to create useful scientific tools by borrowing a preexisting natural entity. One of my favorite things about being a bioengineer is how we are allowed the unique opportunity to be inspired by nature in a realm of synthetic and technological possibilities. Who knew a cocoon-material could one day save your life?

Delivery of non viral gene carriers from sphere templated fibrin scaffolds for sustained transgene expression

Background:

The scaffolds being developed are for replacement of soft tissues. The ideal scaffold is one that has similar mechanical properties as the replaced tissue, and allows for biochemical cues to be integrated to directed the cellular processes and create the necessary organization. Many other scaffolds have controlled rates of degradation which allows for time dependent delivery of growth factors, however special preparation is needed to prevent premature degradation and denaturation. There are some scaffold materials, such as chitosan, collagen, fibrin, or composites that can couple the growth factors to the scaffold via side chain reactions, but these materials generally lack the mechanical strength required for soft tissue replacement, and are broken down too quickly in vivo. Thus, Pun et al decided to deliver the genes necessary for the production of the needed growth factors, instead of the growth factors themselves. Because DNA retains its structural and functional integrity better than proteins and growth factors, pun’s method makes this method compatible with more scaffold preparation methods.

The polymer used, (PEI), forms complexes with DNA to form nanoparticles on the range of 50-200nm in diameter. They are non-immunogenic and are taken up by cells through endocytosis, mediated by electrostatic association between positively charged polyplexes and negatively charged cell surfaces. When the nanoparticle enters the cell, it is hypothesized that it disrupts the endosome by the “proton sponge effect”, which is basically the ability to buffer the endosome and cause its rupture. The DNA will then, by unknown methods, make its way to the nucleus where it then engages in transcription and translation of the desired gene products. The risks associated with viral vectors, such as recombination and mutagenesis are avoided with this nanoparticle approach.

Summary of results:

Pun et al used a three-dimensional, sphere-templated fibrin scaffold to deliver controlled levels of gene delivery vectors. The scaffold was developed by Pun et al such that it has mechanical properties similar to soft tissues. The gene delivery vectors are polymeric nanoparticles, known as polylexes (In this paper, they used polyethylenimine (PEI)). The polyplexes were embedded within the scaffolds using two different methods and the resulting gene delivery and expression profiles are given. One method is embedding the polyplexes within the scaffold, the other method is only coating the polyplexes on the surface. The embedding technique allows for slower sustained delivery, whereas the surface coated approach allowed for much more rapid uptake.

They noticed aggregation of the polyplexes and attempted to pegylate the surfaces of the polyplexes to reduce aggregation. Although it was successful to some extent, this reduced the transfection rate.

The scaffolds tested were prepared by the sphere-templating technique and were fabricated with the dimensions of 2mm thickness by 8mm diameter. The scaffolds were imaged and given an estimated porosity of 74.7 +/- 0.6%, determined by digital volumetric imaging. They mention that further optimization of the porosity is needed.

The expression of the gene delivered VEGF, cell viability relative to a fibrin scaffold control, DNA release from the scaffolds, and long term expression of the delivered hVEGF plasmid were all measured and results are described in the following figures. The cell type used in the experiment were mouse 3T3 fibroblasts.

Figure 1: (A) is a schematic illustration of the sphere template fibrin scaffold production procedure with surface coated or embedded polyplexed or uncomplexed DNA. (B) This is what the distribution of the polyplexes is hypothesized to be like with relation to the fibrin scaffold. The schematic is of a single pore of the sphere template. The blue spheres are the polyplexes and the red spheres are the fibrillar matrix of fibrin.

Figure 2: These graphs demonstrate the release kinetics of the DNA from the scaffold. The diamonds are the surface coated DNA, the surface coated polyplexes of carious N:P ratios are: (circles = 5, triangles = 10, squares = 20). The unfilled diamonds are the embedded DNA, and the unfilled squares are the embedded polyplexes. These graphs demonstrate that the DNA dissociates rapidly from the scaffold, and that the embedded polyplexes adhere strongly to the scaffold.

Figure 3: These graphs depict the long term transgene expression of the hVEGF plasmid, determined by an ELISA assay. (A) Shows the surface coated polyplexes of N:P = 20. These have a peak expression level at day 5, and detectable expression through day 27. (B) Shows cumulative VEGF expression of the same scaffolds in (A). It shows a linear expression through 15 days. (C) Shows the polyplexes embedded with scaffolds. They show peak transfection at day 9. (N:P = 10, gray bars) or at day 7 (N:P = 20, black bars). (D) Shows the cumulative VEGF expression, which shows linear expression through day 29. (N:P = 10, unfilled squares) or through day 21 (N:P = 20, filled squares). The scaffolds with no DNA showed no detectable VEGF expression. (The filled diamonds).

Figure 4: This graph shows the long term cell viability with time on the fibrin scaffolds. The data was normalized to the viability of cells on plain fibrin scaffolds. (filled diamonds) The polyplexes embedded within the scaffolds are (N:P = 20, filled squares). They didn’t show a significant decrease in cell viability. The surface coated polyplexes (N:P = 20, unfilled squares) had a significantly lower viability at days 3, 7, and 14. At day 27, the viability was similar for all of the scaffolds.

Why this paper is pretty awesome:

This non-viral gene delivery method is a nice alternative to the traditional scaffold + growth factor/protein approach. The gene delivery method itself is very nice in that it is non-immunogenic and doesn’t cause recombination and insertion into the cell’s genome like many viral vectors do. Although polymeric nanoparticle gene delivery methods are usually not as effective as other methods, this paper demonstrates that it is sufficient for tissue engineering applications, but I'd like to see it be better.

Attacking Cancer - Fighting with Filaments

Overview

By targeting cytoskeletal filaments such as actin filaments and microtubules, scientists hope to attack cancer cells by disrupting their ability to divide, grow and maintain normal function. Anti-cancer drugs such as taxol and colchicine prevent cancer cells from entering mitosis by greatly stabilizing microtubule structure and thus inhibiting the formation of the mitotic spindle. These drugs can also disrupt the mitotic spindle during the metaphase/anaphase of the cell cycle by destroying microtubule structures, leading the cell into apoptosis. Furthermore, antimitotic compounds such as discodermolide and epothilone can be used to stop the development and proliferation by inhibiting the polymerization of tubulin. Though these treatments are effective, current anti-cancer drugs suffer from specificity; they cannot differentiate between normal and cancerous tissue and therefore cause damage to both.

Therefore, researchers are concerned with finding ways to differentiate between cancer and normal cell dynamics. For example, there are 12 different tubulin isotypes that exist at varying degrees in certain cell types and that also have distinct responses to certain drugs. By finding more information about the differentiation of different cell-types (in this case normal vs. tumor cells), scientists can better develop drugs that target specific isotypes and thus only attack cancerous cells without damaging normal human tissue. Since the actin cytoskeleton is greatly modified in tumor cells due to their abnormal growth and increased ability to divide, scientists have used proteins such as gelsolin and cytochalsin B which have been shown to affect cancerous cells more so than healthy cells. Many actin-targeting compounds are derived from natural marine products such as the lantrunculins.

Why?

Dr. Eschenbach, the Director of the National Cancer Institute stated in September 2004, “…the painful reality is that as we sit here today, one American every minute continues to die from this disease. It remains the disease that Americans fear most because of the suffering and devastation as well as death it brings, and we know that one out of every two men, and one out of every three women in their lifetime will be told they have cancer.” Although many advances in the treatment of cancer have been made such as chemotherapy and radiotherapy, current treatments are extremely painful and have terrible side effects. Chemotherapeutic drugs are cytotoxic drugs that attack cells that are dividing in the body. These drugs are released into the body and damage cancer cells that are dividing uncontrollably anywhere in the body. Since most cells are mature and have stopped dividing they are not susceptible to these effects. Unfortunately healthy cells that are constantly dividing can be damaged as well thus leading to common side effects such as hair loss, lowered blood count and nerve damage. These complications could be reduced and even eliminated with the advancement of specific or localized drug delivery research.

Increasing the Cytotoxicity of NK-like T cells

The goal of this paper was to see whether immunization with different antigens from pancreatic tumor cells improved cytotoxic efficacy of effector cells, such as T cells. Basically, dendritic cells (DC) were pulsed with pancreatic tumor cell-derived RNA and cocultured with NK-like T cells. Tests were run to see if this reversed resistance of target pancreatic cells to the NK-like T cells. Dendritic cells on their own play a small role in immunity to tumor cells by recognizing tumor antigens. This paper was concerned with whether exposure of DC to a host of tumor antigens would increase the destructive capabilities of NK-like T cells cocultured with them.

DC cells were pulsed with the following: no RNA, RNA from pancreatic carcinoma cell lines, poly(A) + RNA, RNA from lymphoma cells, and cationic lipid complex. The results were that the higher the effector : target ratio, the higher the cytotoxic activity of the NK-like T cells. Tests were also run where DC were pulsed with either increasing amounts of total RNA, poly(A) + different amounts of RNA, different amounts of RNA and CA 19-9 (a peptide), different amounts of RNA with or without CA 19-9, or where RNase H was added. All of these tests showed that DC transfected with tumor-derived RNA leads to reversal of resistance of pancreatic tumor cells by triggering cytotoxic effects of NK-like T cells.

This paper was very descriptive and exemplary of the type of research going on now that involves co-culturing along with immunity. I think this is an interesting field with many possible benefits. The next step would be to somehow insert these new NK-like T cells in animals and see if they are effective in vivo. If they are, we are on the road to a potential cure for cancer, which would relieve millions of Americans from the heartache and misery that goes along with this terrible affliction.

Early organogenesis of the kidney

The early organogenesis of the kidney, is a complicated, multi-step process that begins with the development of the ureter. The ureter development is regulated by the surrounding mesenchyme and growth factors which allows the ureter tree structure to branch and proliferate. This ureter growth triggers the endothelialization and morphogenesis of the mesenchyme. The mesenchymal cells will thus differentiate into several types of endothelial cells which will line the endothelium of the kidney, and the mesenchymal cells will undergo morphogenic changes. These two processes propagate each other, as endothelial-mesenchymal cell interaction is a closely intertwined process. The morphological change of the mesenchymal cells starts with loose mesenchyme. This is intertwined with the development and branching of the ureter. The ECM of the mesenchyme changes in composition such that it experiences an increase in several epithelial-type proteins, collagen IV and V, laminin, heparin sulphate proteoglycan, and entactin. DNA synthesis then significantly increases within the mesenchyme. The resulting condensate of cells thus experience increased adhesive capabilities and decreased motility. Most of this condensate ends up gathering at the periphery of the aggregate and serves as a scaffold for the rest of the cells throughout the organogenesis process.

Concurrently, the mesenchymal cells acquire and epithelial, elongated shape within the aggregate. Two slits open within this aggregate, creating the S-shape structure of the early nephron. Endothelial cells migrate into the crevice of the S-shape body and begin the vascularization process. The origin of these endothelial cells is not well understood. Epithelial podocytes and these vascular endothelial cells then contribute to the formation of the glomerular basement membrane, or GBM. Capillary cells from outside then migrate in to the crevice to complete the vascularization process, using the increased concentration of stromal fibronectin to home into the correct location. The key to all of these steps is the formation of the ureter, which serves as an inductor for the rest of the organogensis process. This paper described how without the ureter development, the development of the kidney did not proceed. This chain of events of is still molecular unexplained and some elements are not well understood, but the importance of ureter development is well-established.

The process described in this paper would be essential in any project to engineer an artificial kidney or parts of a kidney. Of interest is the foremost importance of the introduction of the ureter to the whole process. In any method to develop an artificial kidney, the first step would be to introduce a developing ureter structure. This ureter will likely have to be derived from another organism rather than engineered, because the ureter contains within it the “programming” for branching and growth which is not well understood and difficult to emulate in vitro. The introduction of this ureter structure, in theory, should organize and facilitate the remaining development of the organ.

Tissue engineering of blood vessels

In the aging society, diseases related to cardiac and peripheral arteries become the major cause of death to the people. Some patients can be treated with medical care to maintain a living; however, those who have severe arterial problems, they must undergo surgical reconstruction to replace the diseased arteries with some healthy artieres that can have functions. A good substitute for a blood vessel must be strong, compliant, good suture retention, biocompatible, non-toxic, and low thrombogenicity.

There are some options for the substitutes. One option is to use autologous veins from the patient’s self. Mainly the autologous veins come from the saphenous veins of the patient’s legs. However, most patients’ saphenous veins are not suitable for their arterial replacements. Another option is the prosthetic conduit, such as ePTFE or Dacron. However, prosthetic conduits have very high chance of thrombosis in human bodies. Moreover, prosthetic conduits are dead, and they cannot respond to the living environment. They cannot healed when damaged. Thus, it is necessary to develop and grow a living, small-diametered blood vessel and implant it into human bodies. In human-made tissue engineered blood vessel, there are three components: a scaffold, the matrix, and the endothelial cell lining. The scaffold either prosthetic is biological; it provides mechanical supports and constructs the shape of the blood vessel. The matrix helps the endothelial cells to attach. The living endothelial cells develop to healthy, antithrombogenic blood vessel which provides blood flow. Endothelial progenitor cells(EPCs) are seeded on the scaffold in some cases. EPCs Different types of scaffolds are tried: nature scaffolds, permanent synthetic scaffolds, biodegradable synthetic scaffolds. For biodegradable synthetic scaffolds, polymers PGA are used as scaffolds. Then, they are lined up with endothelial cells. After awhile, the grafts look very similar to native arteries morphologically.

The reason I chose this paper is that I am really interested in finding the best substitute for a disease human artery. People eat too well, and a lot of people suffer conditions with ill arteries. This paper gives researchers several pathways into finding a good engineered blood vessel, and it also gives cons and pros for different types of scaffolds. Personally, I think biodegradable synthetic scaffolds will be the most useful and the best materials because they will be degraded after implanted into human bodies, which thus affects humans the least. Also, the materials are not very expensive and can be mass-produced.

Comparison of Scaffolds and Culture Conditions for Tissue Engineering of the Knee Meniscus

The meniscus of the knee is mostly avascular and incapable of regeneration. Thus, meniscal injuries are often permanent. Currently there is no widely accepted treatment. Tissue engineering of the meniscus provides a potential solution.

Agarose is commonly used in tissue engineering because it is easy to mold and seed with cells. Both fibroblasts and chondrocytes have been shown to proliferate on poly(glycolic acid) (PGA). Furthermore, studies using PGA in rotating wall bioreactors have demonstrated increased synthesis of chondrocytes. The authors of this study compared the growth and proliferation of fibrochondrocytes on agarose and PGA in static cultures and rotating wall bioreactors.

Fibrochrondrocytes were harvested from menisci of adult rabbits and seeded on agarose and PGA. Cells were allowed to attach to their respective scaffolds for a week in a static culture. Some scaffolds were then place in a rotating wall bioreactor for another six weeks. Histology was performed to visualize the distribution of collagen and glycosaminoglycan (GAG). Samples were assayed for total GAG and collagen content.

This study showed that fibrochondrocytes grew significantly better in PGA compared to agarose in both the static cultures and rotating wall bioreactors. There was no significant difference between PGA static cultures and PGA rotating wall bioreactors. Cells in PGA also had higher GAG and collagen content than in agarose. From this study, PGA is clearly a more suitable scaffold for tissue engineering of the meniscus.

I found this study interesting because the authors sought to find a suitable scaffold to use before throwing growth factors at the samples. Providing the cells with the appropriate environment and conditions to proliferate seems more important to me than adding a bunch of "extra stuff" that may or may not do anything. The knee undergoes a lot of compressive stress. I think it would be interesting if the authors also compared the cellular response in each scaffold to an applied force. The authors did perform mechanical tests to determine the modulus of each sample, but I think the difference in moduli is due to the material properties of the scaffold.

Neural Tissue Engineering: a Self Organizing Collagen Guidance Conduit

This paper discusses a new method of creating 3-D neural tissue using seeded collagen gels for self-alignment of the tissue. Within the medical community, nerve autografts are the "gold standard," but with the development of this nerve tissue method there is a potential for surgical implantation without removal of other nerve cells that damage donating nerves. Previous studies have shown that collagen gels are an effective way of attaining 3-D tissue cultures, therefore the researchers are applying this knowledge to create nerve cultures that have aligned schwann cells to match true nerve abilities both physically and biologically.

This research used collagen gels (rectangular)seeded with 25,000 schwann cells (prepared from male Fischer rats) per gel. Then dorsal root ganglia (taken from Sprague adult rats') were prepared using DMEM with collagenase. They were later applied to the collagen gels for adherence and growth.

After a 3 day incubation, the gels were washed, and stained with paraformaldehyde to detect betaIII-tubulin, a marker for neurons. Antibodies followed by secondary antibody allowed observation with fluorescence microscope.

The contruction of a device to mimic a nerve began with a silicone tube with eight holes for the collagen seeded gels to be pushed through (like a push pop). This device was inserted into female rat precisely damaged sciatic nerve for in vivo growth studies. If the inserted collagen moved away from the walls of the silicone tube, then self alignment would be considered successful.

The quantification of realignment and growth were performed using transverse and longitudinal sections taken from the insertion site along with the use of fluorescence microscopy.

The results reported showed that the schwann cells within the collagen gell had become aligned with the axis of the tube and DRG neurons extended paralled to the tube walls.

Evaluation of regeneration of a transverse section using the "collagenated tube", no inserted device and empty silicone tube showed that the collagenated had the greatest repairing abilities over time with the most growth.

Longitudinal sections showed the same results with most observed regeneration in the collagenated tubes.

Factors that may influenced results may include variation between rat species, age and sex. The age of the source of DRG may have influeced the potential for regrowth. The change from male to female for the in vivo test may also have affected the amount of regeneration and the direction of the alignment.

This paper presents a good protocal for the development of neural tissue to have correct alignment, essential for nerve communication. This paper was interesting especially after reading previous posts about tissue regeneration. Protocols for tissue development used collagen gels and this further exemplified the potential for 3-D culture. This paper caught my attention because of its attention to detail of the protocol and the future application of this device for treatment of patients with nerve damage such as from car accidents or war injuries.