Brain Implants

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Brain Implants Lars U. Wahlberg NsGene, Inc., Providence, Rhode Island

INTRODUCTION Even though regulatory approved cellular brain implants are not yet available, experimental data strongly support the use of brain implants and tissue-engineering concepts in the treatment of brain disorders and promise new disease modifying medical products for patients. Various cellular implants have already been applied in the clinic in proof-of-concept studies in Parkinson’s disease (PD) [1], Huntington’s disease (HD) [2], Alzheimer’s disease (AD) [3], epilepsy [4] and stroke [5]. PD is a major target for brain implants and is highlighted in this chapter as a disease example to illustrate the tissue-engineering concepts applied to date. Despite the longstanding success of many drugs for PD, such as L-dihydroxyphenylalanine (L-DOPA) therapy, the current treatments of PD do not stop the progressive dopamine neuron dysfunction and cell death. Over time, patients on chronic L-DOPA therapy develop both progressive symptoms and drug-induced side effects and require additional treatment options. In the early 1990s, surgical treatments initially developed during the 1950s, such as the ventrolateral pallidotomy for Parkinsonian rigidity, were rejuvenated [6]. With improvements in imaging and surgical techniques, ablative procedures have yielded excellent results in selected PD patients. Implantable neural stimulators, which inhibit neuronal transmission in local areas by high frequency stimulation, have more recently replaced much of the ablative therapies and can yield good therapeutic results without inducing permanent lesions in the brain [7]. Despite successful applications of neurosurgical procedures for the treatment of PD, these procedures are based on the inhibition or destruction of normal neurons to compensate for the disease damage. They do not address the biology of the underlying disease itself and, albeit successfully applied in many patients, the destruction or inhibition of normal tissue is not an optimal treatment for neurological disorders. There is therefore a need for new treatment strategies that can address the pathology more directly and offer disease modifying effects. Fortunately, the accumulated knowledge of the pathological processes, molecular and cell biology, biomaterials, imaging, and surgical procedures make it now possible to implement disease modifying tissue-engineering concepts to the treatment of PD and other neurological diseases. In many untreatable neurological disorders, the progressive loss of neurons and their associated function is the primary underlying cause for the symptoms of the disease. Therefore, various cell implant strategies have been designed to either replace the neurons or their function or to protect and/or regenerate the function and health of the diseased neurons, or a combination of both (Fig. 62.1). Clinical applications to replace the dopaminergic function in patients with PD have so far utilized primary tissues or cells. Dopaminergic neurons derived from stem cells and more sophisticated tissue-engineered implants have not yet been applied in the clinic but rapid Principles of Tissue Engineering. http://dx.doi.org/10.1016/B978-0-12-398358-9.00062-8 Copyright Ó 2014 Elsevier Inc. All rights reserved.

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FIGURE 62.1 The concepts of cell replacement and protection/regeneration are depicted. A loss of neurons or their function (orange line) occurs with aging but in a disease, such as PD, neuronal loss is accelerated and after a while, the loss is significant enough to cause symptoms of the disease. At this stage, further deterioration could be prevented by protecting the neurons and the system could even be improved by applying regenerative factors to the diseased host cells. After significant loss of neurons, regeneration of the host system is not possible. However, the implantation of cells capable of replacing the function of host cells could be possible. Both approaches have been applied in Parkinson’s disease and a combined treatment of neuroprotection/regeneration with replacement may be achieved in future brain implants.

progress in preclinical efforts is being made. In neuroprotective or restorative strategies, cellular implants secreting either endogenous or engineered growth factors or cytokines have successfully been applied in animal models and even in the clinic for amyotrophic lateral sclerosis (ALS) [8], Huntington’s disease (HD) [9], and Alzheimer’s disease (AD) [10e12]. 1330

This chapter reviews some of the cell replacement and regenerative brain implants applied in the clinic and touches on what may be developed in the future.

CELL REPLACEMENT IMPLANTS Primary tissue implants As mentioned above, oral L-DOPA therapy is the main treatment for PD. L-DOPA is a precursor to dopamine that passes the blood-brain barrier and is mainly taken up by the dopaminergic neurons that convert L-DOPA to dopamine and increase their dopamine production and storage. However, with the progressive loss of dopaminergic neurons, the L-DOPA therapy eventually becomes ineffective and severe fluctuations in the ability to initiate movements occur. Because L-DOPA can increase the production of dopamine and alleviate the symptoms of PD, a reasonable therapeutic approach may be to implant dopamine or L-DOPA secreting cells in the relevant areas of the brain (striatum). Considering this idea, the first clinical transplantation for PD using a cellular brain implant was performed at the Karolinska Hospital in Stockholm, Sweden in the early 1980s [13]. Autologous dopamine-secreting adrenal chromaffin cells were harvested from one of the patient’s adrenal glands and successfully transplanted to the striatum. The procedure was adopted very quickly by the medical community and initial reports indicated good clinical results. With time though, other studies showed poor survival of the cells and minimal positive clinical effects, resulting in the cessation of the treatment [14]. However, the concept of cellular brain implants had made its definite entry into the clinic and many lessons were learned along the way. At about the same time as the first chromaffin cell transplants were made in Stockholm, a promising cell transplantation strategy for PD was developed by Anders Bjo¨rklund and co-workers at Lund University in southern Sweden [4]. They collected discarded aborted fetal

CHAPTER 62 Brain Implants tissue and dissected out the ventral mesencephalon to create cell suspensions containing developing dopaminergic neurons for transplantation experiments. After several years of extensive validation of the concept in animal models, cells were transplanted to the striatum of two PD patients [15]. The first results were safe but relatively unimpressive prompting modifications to various parts of the experimental procedure, and a second pair of patients transplanted about one year later fared much better [16]. These patients showed positive clinical recovery starting about four months after the procedure. Positron emission tomography (PET) data indicated that the grafts survived and took up and secreted dopamine. More than 10 years after the procedure, one of the patients showed persistent graft viability on PET scanning and required only minimal L-DOPA therapy [17,18]. To date, more than 300 patients have been transplanted with fetal ventral mesencephalic tissue at different centers around the world with some encouraging results. However, the lack of suitable donor material, the heterogeneity of the tissues and preparations, and the inability to industrialize the process, have all made it difficult to make standardized medical trials and therapy geared for a large number of patients. Two controlled trials with fetal transplantation showed only minimal efficacy and some patients developed movement side effects (dyskinesias) that appear related to the grafting procedure [19,20]. More recently, it has been observed that grafted fetal neurons may be able to survive for many years after surgery but these same grafted neurons are subject to the same pathological processes that underlie the loss of endogeneous dopaminergic neurons in PD [21]. Therefore, fetal transplantation as a therapy for PD is no longer actively pursued and cell replacement strategies for PD are awaiting alternative cells and implants that lend themselves to a reproducible industrial process applied in well controlled trials. It also should be noted that clinical trials with porcine-derived ventral mesencephalic cells that did lend themselves to a more rigorous industrial process also failed in clinical trials (Pollack, 2001). With concerns regarding the transmission of animal diseases to humans (zoonosis), few if any additional clinical applications of animal-derived cellular brain implants are being contemplated. In fact, the use of animal-derived products in the manufacturing of human brain implants should be avoided to the largest degree possible to minimize safety concerns. Despite disappointing results in the clinical application of both chromaffin and fetal derived primary tissues from both human and pig sources, the experimental work surrounding primary tissue transplantation in PD demonstrated several important points that continue to facilitate the development of tissue-engineered implants for the treatment of PD and other neurological disorders. 1) Allogeneic cells can survive over many years in the brain after a relatively brief initial immunosuppression therapy (18 months) but xenogeneic (porcine) implants do not. 2) Grafted neurons can integrate, function, and interact with the host brain in a physiological and reciprocal manner. 3) Trial designs and outcome measures have been developed that facilitate safety and efficacy measures in the clinic. 4) Surgical techniques have been developed that allow for the safe injection and implantation of cells and tissue-engineered products in the brain. 5) Imaging techniques have been developed to evaluate the implantation and function of the brain implants. 6) Animal models have been developed to translate and scale-up experimental brain implants for the clinic.

Cell line implants As mentioned above, important drawbacks using primary tissues are its limited supply, its heterogeneity, and the difficulty and prohibitive costs to implement the necessary good manufacturing principles (GMP) for harvesting, manipulation, storage, and later use. For example, fetal transplantation experiments for PD required fresh tissue from 4e8 donors resulting in procedural difficulties and poor quality control and may help explain the poor

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PART 15 Nervous System and variable efficacy outcomes in controlled trials. Therefore, the ability to expand and store cells in cell banks is paramount to creating allogeneic cell alternatives to primary tissue sources. The expansion of normal and genetically unmodified cells derived from tissue donations can give rise to expandable primary cell lines potentially useful for brain implantation with or without further manipulation. The primary cell lines normally retain a limited number of cell cycles but allow for the proliferation of enough cells to transplant hundreds to thousands of patients from a single donor. Cultured primary cells can be expanded while retaining normal genotypes and phenotypes with normal contact inhibition and differentiation behaviors. These cells are therefore relatively safe to use, and the formation of tumors or other abnormal behaviors are relatively unlikely. Retinal pigmented epithelial (RPE) cells derived from the retinas of organ donors can make primary cell lines with limited expansion capacity and have been evaluated in clinical trials for PD. The RPE cells secrete L-DOPA and are thought to function by increasing the intrastriatal L-DOPA concentration and subsequent conversion to dopamine by residual dopaminergic nerve endings and glia. These cells are grown and transplanted on gelatin microcarriers to improve survival and prevent immune rejection. A report on a clinical pilot trial showed that these implants were well tolerated and safe [22]. However, a recently completed Phase II trial failed to reveal any evidence of efficacy [23] and earlier published autopsy results demonstrated poor cell survival at six months [24].

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A more versatile source of expandable primary cell lines capable of making various replacement neurons or glia are mitogen responsive human neural progenitor/stem cell cultures that can be isolated from various regions of aborted and adult human central nervous system (CNS) tissues and expanded for more than one year in vitro. These cells are often grown as ‘neurospheres’ and can be expanded in defined and animal-free media containing growth factors [25]. These cells can form the three major phenotypes of the nervous system (neurons, astrocytes, and oligodendrocytes) in vitro and in vivo and show excellent survival without the formation of tumors in vivo. These human neural stem cell-containing cultures have been transplanted to various regions in animals and survive, integrate, migrate, differentiate, extend neurites, and arborize [26]. Even though the cells tend to retain the markers consistent with the anatomical region from which they were isolated [27], the cells can be manipulated with epigenetic and genetic factors to make specific cellular subtypes potentially useful for cell replacement implants. For a PD application, relevant nigral dopaminergic neurons need to be generated from these cells. However, so far only dopaminergic neurons with limited differentiation have been generated [28] although improvements are consistently being made [29]. In addition to neurospheres, adherent primary cell lines of neurogenic glia have been made from both mouse and human developing neural tissues that display a glial phenotype during expansion but are capable of generating neurons during differentiating conditions [30]. Similarly, so called NS cells, expressing glia and stem cell markers have been derived from ES cells and appear to differentiate into similar neural phenotypes as the adherent glia and neurosphere cultures [31]. The adherent cultures may have an advantage over neurospheres from an industrial point of view as adherent cells can be readily cloned, expanded, and have shown to retain their phenotype during expansion without differentiating into a heterogeneous mixture of progenitors. It is currently unclear if neurospheres or adherent NS cells can be made into dopaminergic neurons suitable for replacement therapy in PD. Reportedly, clinical applications in Batten’s disease with unmanipulated human neurosphere cell lines are currently being explored [32]. In general, all primary cell lines derived from tissue stem cells have a large but limited expansion potential and show senescence [33,34]. This may be due to the successive loss of immortal stem cells through the asymmetric division into progenitors (as seen in neurospheres) or alternatively, the stem cells themselves have a limit to their proliferation.

CHAPTER 62 Brain Implants Both mouse and human ES cells defy the normal senescence of primary cells and can be expanded from a single clone indefinitely without losing pluripotentiality [35]. From an industrial and tissue-engineering perspective, this feature is extremely attractive as a single donation could give rise to a cell line source with the capacity to make all organs of the body in unlimited numbers. One major drawback of ES cell-derived products, however, is that the ES cell itself cannot be implanted but needs to undergo the relevant development in vitro to make suitable cells for transplantation, e.g., dopaminergic neurons for PD or islet cells for diabetes mellitus. As it is difficult to make pure cultures without retaining one or more undifferentiated ES cell, both the risk of heterogeneous cell preparations and the risk of tumorigenesis need to be overcome to develop ES cell-derived brain implants [36]. For neural applications, the recently described NS cell may become a progenitor of choice for brain implant products as it is restricted to the neural lineage and can be expanded as a clonal cell line. It is currently unclear if the undifferentiated NS cell is immortal like its parental ES cell or if it displays senescence as its neurosphere counterpart. Cell replacement strategies using cell lines need to employ differentiation or selection methods in order to make the relevant replacement cell, e.g., dopaminergic neurons for a PD application. The generation of functioning human dopaminergic neurons in vivo akin to those derived from primary VM tissues has been difficult to achieve. Although neurons with the dopaminergic machinery can be made from both growth factor expanded human neurospheres and genetically immortalized committed dopaminergic neuroblasts, it has been difficult to achieve survival and function in vivo [28]. Interestingly, even though ES cells would hypothetically need more steps to be differentiated into dopaminergic neurons for a PD application, relatively short-step protocols that use developmental signals involved in the rostrocaudal and ventrodorsal specification of the midbrain can push mouse ES cells into functional dopaminergic neurons in a rat model of PD [37,38]. Studies have also identified important transcription signals involving the Lmx1a and MSX homeobox genes that when overexpressed in ES cells under the nestin promoter can yield dopaminergic neurons with markers consistent with substantia nigra neurons [39]. Transplantation of these dopaminergic neurons in a rat model of PD yields excellent survival, neurite extension, and function consistent with results from primary VM tissues. However, similar to the human ES cells [36], these cells also form tumors in vivo. These findings may pave the way forward to make relevant ‘nigral’ dopaminergic neurons from human ES cells and neural stem cell cultures in the not too distant future but also shows the need for adult and fetal derived neural stem cell sources that do not form tumors.

CELL PROTECTION AND REGENERATION IMPLANTS The use of primary tissues or cell lines in cell replacement approaches are aimed at making transplantable mimics of the cells lost in the disease process. In PD, the use of chromaffin cells, dissected developing ventral mesencephalon, RPE cells, or stem cells have all been aimed at replacing or augmenting the dopaminergic function. However, primary tissues, cell lines, and genetically modified cells also produce secreted factors that can influence the nearby host or transplanted cells in potentially beneficial ways. Several growth factors are endogenously made by cells including fibroblast growth factors, transforming growth factors, and interleukins that can have neuroprotective and regenerative effects on nearby nerve cells. Custom therapeutic cell lines can also be made by genetic engineering to secrete specific growth factors such as nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) that when implanted in relevant anatomical areas can affect specific neuronal populations in neuroprotective and regenerative ways (ex vivo gene therapy). To protect the transplanted cells from immune rejection and to allow for the retrieval of the therapeutic cell implants an encapsulated device can be used.

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Cell implants secreting endogenous factors A carcinoma cell line derived from a human testicular teratocarcinoma isolated from a metastasis in a patient has been used in clinical applications to treat the neurological sequelae of stroke [40]. This immortal cell line is pluripotent and can be induced to stop dividing and to differentiate into a neuronal phenotype using retinoic acid. A preparation of this cell line was investigated in the clinic for the treatment of ischemic stroke based on animal data suggesting that the post-injury transplantation of this cell line into an infarcted area can improve recovery. The mechanisms surrounding this effect are unclear but are likely related to beneficial factors released from the cells. In a study in patients with lacunar stroke in a randomized controlled Phase II trials at the University of Pittsburgh, USA the therapy with this cell line failed to meet the efficacy endpoints. The transplantation of a cell line derived from a human cancer has obvious risks associated with it. Importantly, the approval of this trial demonstrates that cell transplantation for severe neurological disorders is seen as a reasonable strategy by the regulatory agency, as long as safety and some efficacy can be demonstrated in animal models.

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Other groups are investigating the transplantation of immortal cell lines but are using genetic engineering to immortalize cells. Advances in genetic engineering have made it possible to extend the number of doublings a primary cell line can go through by inserting various oncogenes and cell cycle regulators. This allows for the selection, clonal expansion, and banking of a large number of cells. Besides the genetic modification, these cells retain otherwise normal genotypic characteristics. ReNeuron, a British biotechnology company, has made immortal human neural stem cells that have shown regenerative effects in stroke models [41] and have initiated clinical trials in stroke patients [42]. Similarly, StemCells Inc., is using growth factor expanded human neurosphere cell lines in a strategy to treat Batten’s disease, a rare neurometabolic disorder, with the idea that the endogenous enzymes and factors made by the stem cells will have a therapeutic effect [32,42]. Lastly, implanted autologous mesenchymal stem cells are being studied in clinical trials for stroke [43] and potentially in neurodegenerative disorders [44]. The use of non-specific neuroprotective and regenerative strategies based on the implantation of cells with unclear mechanisms may pose regulatory problems as the risk benefit analyses become difficult to make. For example, even though positive results were inferred from the published trial with the human teratocarcinoma derived cell line in stroke, the clinical data were not convincing enough to continue clinical development [45]. In this trial, no significant adverse events or tumors were reported but, if it they had occurred, a major set-back for tissue-engineered brain implants could have been the result. The risk benefit analysis is often difficult and the predictive value and scale-up issues by the use of animal models are not straight forward. The regulatory agencies have therefore a real dilemma and, similar to the setbacks experienced in the field of gene therapy, a push to do clinical trials with poorly characterized cell preparations and mechanisms in patients desperate for a treatment may cause significant adverse events that can create set-backs for the whole field of tissue engineering. On the other hand, a too restrictive regulatory body may make the hurdle of bringing potentially beneficial but complex tissue-engineered products into the clinic too costly and difficult. These regulatory issues are hard to resolve but as experience with cell-containing implants build, it is likely that the decision making and risk benefit analyses will improve. Some of the clinical trials using primary autologous cells such as hematopoietic or mesenchymal stem cells derived from the bone marrow can also by-pass regulatory scrutiny and only need approval by a local ethics committee. Unfortunately, this has led to the initiation of clinical trials based on very little evidence of preclinical beneficial effects causing potentially false hopes, personal expenses, and potentially harmful side effects to patients desperate for therapy.

Cell implants secreting engineered factors (ex vivo gene therapy) As PD involves a slow and progressive degeneration of dopaminergic neurons a protective and/or regenerative strategy could be applied. Many protein factors have been shown to

CHAPTER 62 Brain Implants protect fetal dopaminergic neurons both in vitro and in vivo and one of the most powerful factors is glial cell line-derived neurotrophic factor (GDNF) [46]. This factor promotes the survival (neuroprotective effect) and neurite extension (regenerative effect) of dopaminergic neurons both in vitro and in vivo. Based on positive animal data, GDNF has been tried in humans in two separate randomized clinical trials [47,48]. The first trial used monthly intracerebroventricular bolus injections of GDNF and failed to meet both safety and efficacy endpoints. The second trial employed intrastriatal infusion of GDNF and albeit safe, did not meet the efficacy endpoints. This is an unfortunate outcome for patients suffering from PD and critics of the trial have indicated that inconsistencies in catheter design and other parameters may explain the poor results [49]. From both the clinical and animal data, it appears necessary to deliver the GDNF in low but chronic doses to the dopaminergic nerve endings within the striatum in order to have a regenerative effect. It is currently the view of many that implantation of genetically modified cells, encapsulated cells, or the use of viral vectors may be better at delivering GDNF than utilizing available pumps and catheters. Safety concerns with unregulated and unstoppable gene therapeutic approaches may favor the use of a tissue-engineered product based on an implantable and retrievable encapsulated cell biodelivery of GDNF. Of note, however, Ceregene, inc. has completed a Phase II clinical trial demonstrating safety of AAV-mediated delivery of Neurturin (a GDNF analog) in PD patients [50] and has recently initiated a follow on trial. It is beyond the scope of this chapter to expand on the topic of ex vivo and in vivo gene therapy but it is important to mention that the delivery of growth factors to the brain via implants is an important cornerstone of tissueengineering strategies for brain repair.

Encapsulated cell implants The implantation of naked cells has the advantage of allowing for migration, integration and the formation of neurites and synapses in replacement strategies. The migration and homing mechanisms that neural stem cells display in models of stroke and glioma may also be utilized to deliver regenerative or tumoricidal agents respectively in genetically modified cells. However, naked cells cannot readily be removed and if a potent protein factor is being delivered to the brain, the inability to stop the treatment may pose a problem if untoward effects are noted or if the regenerative treatment is only needed for a limited amount of time. A device containing encapsulated cells that secrete the factor combines the advantages of cell and gene therapy with that of the safety of a retrievable device. One of the types of encapsulated cell devices is depicted in Fig. 62.2 and consists of a hollow fiber membrane that surrounds a core of cells seeded on a polymer matrix that in turn is attached to a tether. These encapsulated cell implants are true tissue-engineered devices that combine genetically modified cell lines with artificial scaffolding enclosed behind an immunoprotective membrane. The polymeric membrane excludes larger molecules and cells but allows for the bi-directional passage of nutrients and transgene products. The encapsulated cells can thus be protected from immune rejection, making allogeneic or even xenogenic transplantation possible and immunosuppressive therapy unnecessary. The host is also protected from the implanted genetically modified cells and the risks of gene transfer or tumor formation are greatly diminished. The tether allows for handling, implantation, and removal. These devices can be implanted intraparenchymally, intracerebroventricularly, or intrathecally depending on the application. Cellular survival and continuous production of factors have been demonstrated for at least 12 months in the brain allowing for longterm delivery of therapeutic factors [11,51]. Encapsulated devices secreting GDNF have been studied in rodent models for PD and have shown both neuroprotective and neuroregenerative effects on dopaminergic cells [52]. A recent Phase I trial in Alzheimer’s patients was completed demonstrating safe and targeted delivery of NGF to the brain using encapsulated, genetically modified ARPE-19 cells. The cells survived for up to12 months with no evidence of inflammation or device displacement [10,11].

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FIGURE 62.2 A schematic of the implantation of four encapsulated cell implants in the human basal cholinergic forebrain is depicted. The tip of the implant houses a human immortal cell line engineered to secrete the therapeutic protein (in this particular application NGF). The cell line is grown on a three dimensional synthetic scaffolding and the cellular core receives nutrients and oxygen from the surrounding brain interstitial fluid via the semipermeable membrane. In turn, the therapeutic protein can diffuse from the implant tip in a few millimeter wide radius into the surrounding brain at the implant target and the associated diseased neurons. The membrane protects the engineered cells from immune rejection and the entire device can be removed or replaced through the use of the tether anchored beneath the skin at the skull bur hole level. The sections are stained with hematoxylin and eosin. The implant measures approximately 1 mm in diameter.

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Other investigators have published data on using encapsulated choroid plexus cells in the treatment of Huntington’s disease [53] and PD [54]. These cells are reportedly therapeutic by secreting various endogenous factors that have neuroprotective and regenerative effects. The application of the choroid plexus cells in this study was made with an injectable micro-encapsulated cell configuration. In this setting, the encapsulation provided immunoprotection for the porcine-derived primary choroids plexus cells. Unlike the macro-encapsulation described above, these devices would not be retrievable and may be less suited for applications in which the treatment may need to be stopped.

Controlled release implants Acellular synthetic polymeric brain implants that are able to deliver protein factors or other drugs to the CNS have also been developed [55,56]. These systems normally release drugs by degradation- or diffusion-based mechanisms over an extended time (weeks) but cannot sustain release over a long time (months), which is possible with cellular-based systems. Appropriately designed, polymeric controlled release devices have several possible applications and could for example support the survival and integration of transplanted cells. Furthermore, a polymeric system can support the sequential release of growth factors that may be necessary to fully support the stepwise differentiation of immature cells. This concept could become applicable to transplanted neural stem cells that may lack important embryonic developmental signals in the adult brain.

COMBINED REPLACEMENT AND REGENERATION IMPLANTS From a tissue engineering point of view, the future goal is to make replacement organs for the body that can take over the function of a failed organ or structure in an anatomically and physiologically correct manner. And even though it would be difficult if not impossible to make entirely new brains, it should become possible to not only replace cells but also to make new axonal pathways and restore the correct connections.

CHAPTER 62 Brain Implants The transplantation of fetal dopaminergic cells to the striatum is called heterotopic transplantation. This means that the dopaminergic cells are transplanted into an anatomical region different from their normal location, which is the substantia nigra. The heterotopic implantation of dopaminergic neurons may result in the loss of important normal innervation and feedback loops. Many transplants for PD may thus only work as simple cellular pumps that increase the striatal dopamine levels. Although simplicity is desired, an ultimate strategy to treat PD could be to transplant the dopaminergic neurons to their anatomically correct position (homotopic), regenerate the nigrostriatal axonal pathway, and induce terminal sprouting and innervation of the striatal target neurons. This would regenerate the appropriate connections and represent a more physiologic strategy. An initial approach may be to provide survival factors to the implanted cells. Even in the most optimal VM grafts applied heterotopically in PD, the total fraction of surviving dopaminergic neurons was only about 10e20% [57]. This required a large number of donors (4e8) to assure enough surviving cells for a clinical effect. The combination of VM grafts with a neuroprotective and regenerative effect of GDNF is therefore a logical idea. Experimental data indeed show that the application of GDNF delivered by encapsulated cells in combination with either rat or human VM grafts increase both the survival, neurite extension, and innervation of the striatum in a rat model of PD [58,59]. Similarly, it would be expected that GDNF would have survival and regenerative effects on dopaminergic cells derived from ES or other stem cells when placed in vivo. A combined approach with dopaminergic grafts and encapsulated cell implants secreting GDNF may therefore be contemplated in future transplantation studies in PD. A large challenge for tissue-engineering approaches in the treatment of neurological disorders is the regeneration of axonal pathways. Axons between the cell bodies and their targets often extend for several centimeters in the brain and close to one meter between the brain and the lumbar spinal cord neurons in an adult. Compared to the relatively short distances that the axons had to grow during development, the regeneration in the adult may pose a particular challenge. Fortunately, science has made progress in this area and what was though impossible only a few years ago now seems more feasible. Several molecules are now known to both promote and guide axonal outgrowth. As mentioned, GDNF is a strong promoter of axonal outgrowth of dopaminergic neurons. In addition, certain extracellular matrix (ECM) proteins, such as laminin, can guide axonal outgrowth [60] and extensive nigrostriatal reconstruction has been accomplished using bridges of striatal tissues in combination with fetal mesencephalic grafts placed in the substantia nigra [61]. The finding that the central myelin and glial scars are inhibitory to axonal outgrowth, has led to the identification of various inhibitory molecules that can be manipulated in various ways with inhibitors and enzymes [62]. From a tissue engineering point of view, the combination of replacement cells with regeneration channels or scaffoldings capable of releasing survival and neurite promoting factors and coated with molecules that facilitate axonal outgrowth, may thus become a future reality. In combination with nanotechnology, synthetic bridges can be made that promote extensive fiber regeneration and functional restoration [63]. Even though there mounting data show that axonal bridges can improve axonal growth in animal models, these methods have not been applied to humans. As the cellular building blocks become better refined, it is likely that more ‘true’ tissue-engineered brain implants will enter the clinic. These types of implants could have great potential use for regeneration in many areas of the CNS, particularly the spinal cord.

DISEASE TARGETS FOR BRAIN IMPLANTS As mentioned, PD has been a major target for cellular brain implants. However, many other neurological disorders should become amenable to tissue-engineered implants.

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PART 15 Nervous System In Huntington’s disease (HD), several neuronal populations slowly degenerate and cause the clinical signs of choreiform movements and progressive dementia. HD is inherited as an autosomal dominant disease and the responsible mutation has been located to chromosome 4. Carriers of the disease can therefore be screened for and identified before the onset of symptoms. This makes a neuroprotective strategy for HD an attractive possibility, where the delivery of neurotrophic factors could prolong the symptom-free interval. One such factor is CNTF that protects striatal neurons in both rodent and non-human primate models of HD [64]. Considering these results, a small clinical proof-of-concept trial using an intracerebroventricularly placed encapsulated cell device secreting CNTF was completed [9]. The study indicated that the placement was safe and would warrant additional trials. However, data also suggested that improved devices and an intrastriatal implantation approach may improve the efficacy. This approach may be taken in future trials. Cell replacement strategies have also been tried in HD [2]. Primary fetal striatal tissue transplantation for HD has been performed at a handful of centers in the world. Long-term follow up has described mild improvements in some of the implanted patients [65]. Because the number of implanted patients is few, it is currently difficult to draw any major conclusions regarding the clinical efficacy of transplantation for HD. One possible advantage over PD is that the transplantation for HD involves homotopic implantation, which at least theoretically should allow for the differentiation of the fetal tissue using normal environmental cues. However, in HD, multiple sets of neuronal populations degenerate including both cortical and striatal neurons. The homotopic transplantation for HD may thus require more extensive regeneration of axonal pathways than in PD.

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Other diseases that could be amenable to the implantation of cells within the brain are the myelin disorders. Animal experiments have shown the ability of neural tissue, purified oligodendrocytes, oligodendrocyte precursors, immortalized glial cells, and neural stem cells to remyelinate areas of demyelination [66]. One substitute for oligodendrocytes may be to transplant growth factor expanded Schwann cell (the myelinating cell of the PNS). This is based on the fact that patients with CNS myelin disorders do not display demyelination of the peripheral nervous system. In fact, remyelination of central axons may spontaneously occur by ingrowth of Schwann cells from the periphery. An intriguing therapeutic possibility is therefore to grow Schwann cells from a nerve biopsy and expand these cells in culture. In turn, these cells could be transplanted into demyelinated areas in the same patient. Physiologically, this may not be the best strategy as Schwann cells only myelinate single axons, whereas an oligodendrocytes myelinate multiple axons. However, animal data indicate that, for a limited volume, Schwann cells can remyelinate and restore function to central demyelinated areas. Based on the animal data, a small clinical study was performed. Reportedly, the Schwann cell transplants did not survive but the procedure appears to be safe [67]. It is currently unclear if new trials with Schwann cells are to be expected. One of the most common neurological disorders is epilepsy that affects about 1e2% of the population. Epilepsy is characterized by recurrent abnormal electrical discharges in the brain affecting subparts of the brain or generalizing to deeper parts in the brain resulting in unconsciousness. A subgroup of these patients has temporal lobe epilepsy that is generated by a loss of neurons and an imbalance of inhibitory and excitatory neurotransmitters in the hippocampal formation. In medically intractable cases, this disease can sometimes be treated surgically with the removal of the medial hippocampus and the abnormal area. This procedure eliminates or reduces the frequency of seizures in selected patients but involves a major surgical procedure and the ablation of normal tissue. A less invasive procedure may be to implant inhibitory cells in the seizure focus that would raise the seizure threshold [4]. This idea is supported by animal experimentation data that indicate that locus coeruleus grafts and the local delivery of inhibitory substances such as GABA can increase the seizure threshold.

CHAPTER 62 Brain Implants However, with the lack of suitable cells, such as GABA producing cells, transplantation for focal epilepsy has not been as extensively studied as for some of the aforementioned disease targets. Recent preclinical studies have suggested that interneuron precursor cells derived from the medial ganglionic eminence may be a source of inhibitory GABAergic neurons perhaps providing a useful source of transplantable cells for epilepsy [68]. Other disease indications that may benefit from brain implant strategies include stroke, brain injury from trauma, Alzheimer’s disease, and rare disorders such as cerebellar degeneration and inherited metabolic disorders. Besides the brain, the spinal cord and retina are potential targets for similar approaches.

SURGICAL CONSIDERATIONS The surgical implantation of most brain implants involves the use of stereotactic techniques. The stereotactic method (stereotaxis) in brain surgery was established in the beginning of this century and is now well established in neurosurgical practice [6]. It involves attaching a rigid frame (stereotactic frame) to the skull followed by imaging such as MRI. Attached markers (fiducials) create a three-dimensional coordinate system in which any point in the brain can be defined and related to the frame with high precision. In the operating room, the markers used during imaging are replaced with holders that guide the instruments. It is a relatively simple neurosurgical procedure often done under local anesthesia and mild sedation. The procedure is therefore safe and relatively painless. The patients are usually discharged from the hospital after an overnight observation.

CONCLUSIONS In this chapter, various brain implants have been described that may have potential to treat PD and other neurological disorders using tissue-engineering strategies. Most of the current literature describes the transplantation of various primary cells such as fetal tissue. Tissueengineering principles and cell lines have only more recently been introduced. Applications using growth factor support, genetic engineering, scaffolds, extracellular matrices, and encapsulation have all been able to improve the survival and function of the brain implant. The ultimate implants are yet to be developed and may combine stem cells, genetically modified cells, controlled delivery devices, axonal bridges, scaffolds, and encapsulated cells.

Acknowledgment The author acknowledges and appreciates the review of the manuscript by Dr. Dwaine Emerich.

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