Table of contents :

• implantation of freshly isolated or cultured cells : individual cells or small cellular aggregates from the patient or a donor are either injected into the damaged tissue directly or are combined with a degradable scaffold in vitro and then implanted.
• in situ tissue regeneration : a scaffold implanted directly into the injured tissue stimulates the body's own cells to promote local tissue repair. The construction of stable blood vessels is a fundamental challenge for tissue engineering in regenerative medicine. Engineered blood vessels have often been found to be immature and unstable^ref
• genes that enhance the survival and/or proliferation of vascular cells - endothelial cells and mural cells - can be introduced to extend the lifespan of the engineered vessels^{ref1, ref2, ref3}, but these may prove to be oncogenic.
• a network of blood vessels stable and functional for one year in vivo can be formed in mice by co-implantation of human umbilical-vein endothelial cells (HUVECs) and 10T1/2 mesenchymal precursor cells in a 3D fibronectin-type I collagen gel^ref. The 10T1/2 cells differentiate into mural cells though heterotypic interaction with endothelial cells^{ref1, ref2, ref3}. To permit continuous observation of the engineered vascular networks in vivo, a 3D construct was implanted in mice bearing transparent "windows"^ref and the gene for EGFP was introduced in order to track the implanted HUVECs^ref. The likely existence of endothelial and of smooth-muscle progenitor cells in adults^{ref1, ref2} indicates that these cells might serve as a source of autologous cells for engineering blood vessels by using the approach described here.
• implantation of tissues assembled in vitro from cells and scaffolds (tissue engineering) : a complete 3D tissue is grown using patient or donor cells and a scaffold, and then is implanted once it has reached "maturity"

• implants : without restablishing vascular and nervous connections
• avascular graft / white graft : a graft of tissue in which not even transient vascularization is achieved
• corneal allograft
• full-thickness corneal allograft (lasting 30-40')
• penetrating graft
• lamellar allograft preserving Descement's membrane and corneal endothelium in recipient's cornea (lasting 2 hr)
As the cornea is not vascularized transplant rejection occurs in less than 5% of recipients and can be prevented with topical corticosteroids. Rejection is more frequent when cornea has become vascularized (eg. in herpetic keratitis). Quality directly relates with number of endothelial cells. Cornea can be included in a conservation medium (eg Optisal^®) for up to 1 week or in a culture medium for up to 1 month. A punch is used to cut a circular fragment from the donor cornea and a suction mechanical trapane is used to remove a same fragment from the recipient's cornea. The suture is left for 1 year^ref.
• dental implant : a prosthetic device of alloplastic material implanted into the oral tissues beneath the mucosal or periosteal layer or within the bone to provide support and retention to a partial or complete denture.
• endosseous implant / endosteal implant : a dental implant made of metal or sometimes ceramic or polymeric material, consisting of a blade, screw, pin, or vent, inserted into the jaw bone through the alveolar or basal bone, either directly or through the root canal and apex of a tooth, with a post protruding through the mucoperiosteum into the oral cavity to serve as an abutment for dentures or orthodontic appliances, or to serve in fracture fixation.
• endodontic implant : a metallic implant extending through the root canal of a tooth into the periapical bone structure, thereby lengthening the root of a pulpless tooth.
• intraperiosteal implant : a frame that conforms to the shape of the jawbone and is implanted beneath the outer or fibrous layer of the periosteum so that it rests firmly on the bone, with a post protruding into the oral cavity to serve as an abutment for dentures; used most commonly for upper fixed partial dentures and in the treatment of cleft palate.
• osseointegrated implant : an endosseous implant containing pores into which osteoblasts and supporting connective tissue can migrate. Metallic, ceramic, and polymeric materials have been used.
• Brânemark implant : a type of osseointegrated implant consisting of a two-stage system of titanium screws
• subperiosteal implant : a dental implant consisting of a metal frame implanted under the periosteum and firmly bound by the mucoperiosteum, resting on the jaw bone, with a post protruding into the oral cavity; used most commonly as an abutment for upper fixed partial dentures and in the treatment of cleft palate.
• transmandibular implant : a dental implant consisting of baseplate, cortical screws, threaded posts, and a superstructure that attaches to a denture, inserted via submandibular incision and fixed to the symphyseal border. The implant transverses the mandible, bears the denture directly, and is designed for patients with severe mandibular alveolar atrophy.
• Bosker implant : a common type of transmandibular implant.
• transosteal implant : a dental implant consisting of a bolt that crosses the mandible and is secured in place by a nut; a post protrudes into the oral cavity and serves as an abutment for dentures or orthodontic appliances.
• transplantations and transfusions : reestablished vascular and nervous connections

Donor and recipient : warm ischemia time (from arterial clamping to placement into cold organ perfusion solution) => cold ischemia time (CIT) (from placement into cold perfusion solution to beginning of vascular anastomoses in the recipient) => second warm ischemia (from beginning of vascular anastomoses to arterial declamping in the recipient)
• autotransplantation / autotransplant / autograft / autochthonous, autogenous or autologous graft (if the donor is the recipient itself : e.g. bone marrow, skin, ovary, spleen, parathyroid, fatty tissue, ...)
• syngeneic transplantation / syngraft / isograft and isogeneic, isologous, or syngeneic graft / isotransplantation : a graft between genetically identical individuals. Typically, syngrafts are grafts between monozygotic twins, between animals of a single highly inbred strain, or between F₁ hybrids produced by crossing inbred strains
• allogeneic transplantation / allotransplantation / allotransplant / allograft / homograft / homologous or homoplastic graft (if the donor belong to Homo sapiens species) : transplantation of an allograft; the 3 types are
• cadaveric donor transplantation : allogeneic transplantation of an organ or tissue from a cadaver
• grafts from heart-beating donors (HBDs) : heart => lungs => liver => pancreas => kidneys

Techniques :
• no touch fast technique (sequential surgical equipes) : median sternotomy => preparation of arteries; median xiphopubic laparotomy (also transverse incision in obese patients); preparation of inferior vena cava => exploration of abdominal cavity => cannulation of subrenal abdominal aorta and inferior vena cava in hemodynamically unstable patients; isolation of choledocus, hepatic artery, gastroduodenal, right gastric artery and subhepatic vein for liver explantation => ligation of inferior mesenteric artery => isolation and cannulation of inferior mesenteric vein for portal perfusion => cholecystotomy and lavage of bile from abdominal cavity with NS to prevent autolysis and liver damage => heparinization (3 mg/kg i.v.) (when the heart is explanted, phenothiazines cannot be administered) => placement of angiostat over supradiaphragmatic aorta and superior vena cava => discontinuation of systemic i.v. fluid infusion => cold perfusion with EW through subdiaphragmatic aorta (and, if portal vein is perfused, also through inferior mesenteric vein) => cardiectomy and pneumonectomy. Cooling of abdominal viscera can be enhanced by introduction of sterile ice into the abdominal cavity => ligation of right gastric and gastroduodenal artery and isolation of common hepatic artery until celiac trunk => pancreatectomy (the choledocus is sutured at the upper border of pancreas) and hepatectomy together with celiac trunk. Nephrectomy is performed at last. Incision of right parietocolic pouch => medial mobilization of ascending colon and terminal ilium => distal ligation of superior mesenteric artery => aortic clampage and cold perfusion => mobilization of asceding colon and sigma by incision of left parietocolic pouch along Toldt white line => ligation of inferior mesenteric vessels => kydneys are made free from retroperitoneum and psoas but still into Gerota's capsule (which is then removed at the bench). Ureters are sectioned just above the cupule of the urinary bladder to prevent excessive traction. Periureteral adipose tissue has to be preserved in the golden triangle (from distal ureter to lower renal pole) as the ureteral artery (emerging from the lower border of the renal artery) is the only one left for vascularization of ureter. For en-bloc explantation the left kidney and ureter are passed through an incision in the mesocolon. Aorta and inferior vena cava are sectioned distally to perfusion cannulas after looking for eventual lower polar arteries emerging from common iliac artery. The large vessels (still cannulated) and ureters are moved upward  => the posterior lumbar vessels are ligated and dissected => section of aorta and inferior vena cava just below diaphragm. At the bench the left renal vein (longer) is sectioned, while the right renal vein (shorter) is left attached to the inferior vena cava (sectioned just above the entry of the right renal vein and then used to enlenghten the right renal vein itself). The posterior face of the aorta is incised longitudinally between emergence of lumbar arteries beginnig from proximal aorta and respecting eventual anomal renal arteries. An aortic patch including the celiac trunk and the superior mesenteric artery is cut; a trasverse incision between the ostium of the superior mesenteric artery and the ostium of the renal arteries; a second transverse incision is practiced over aorta downstream of renal arteries to leave a wide patch around them. The 2 kidneys are then separated by a longitudinal incision in the anterior wall of the aorta between the ostium of the right and left renal arteries. The iliac arteries and veins are explanted en bloc leaving bifurcations intact : even the 2 larger saphena veins are explanted sectioning them 2 cm below the knee, as they could be useful for eventual successive vascular reconstructions at the bench in renal or other organ transplantation.

Complications :
• excessive resection of subhepatic vena cava and damage to right renal vein
• injury to eventual multiple renal vessels emerging from iliac arteries
• too wide resection of aortic patch by the liver surgeon damaging right renal artery
• total abdominal evisceration (TAE) (Nakazato : 2 surgeons required) : preliminary dissection performed by the surgeon and physician's assistant averaged 30 to 45 minutes before aortic cross-clamping. Removal of all abdominal organs (liver, kidneys, pancreas, bowel) en bloc averaged 16 to 24 minutes after aortic cross-clamping, depending on the speed of the thoracic procurement.^ref
• grafts from non-heart beating donors (NHBDs) preserving some brainstem functions

Techinique : a catheter is introduced in the femoral artery to make a fast cold perfusion contemporaneously administering heparin. After clamping of thoracic aorta, the kidneys are rapidly removed en bloc and separated at the bench
• [brephoplastic graft : the transplantation of tissue from an embryo or newborn to an adult animal]
In the early 1960s, cadaveric donations were thought to be impractical and impossible. Living donors were the only available source of organs for transplantation. At Massachusetts General Hospital, a liver was harvested from a police officer whose heart was beating but whose brain was deemed dead. This seminal event led to the development of the concept of brain death as death, rather than the cessation of circulation, which previously defined death. The concept of brain death greatly increased the number of organs available for donation and improved the preservation of harvested organs. Once the concept of brain death was established, a system for organ procurement was founded to ensure the quality and availability of organs as efficiently as possible.
Contraindications : well-differentiated thyroid papillary carcinomas > 1 cm in diameter, as well as follicular and medullary carcinomas (regardless their size and or clinical staging), present absolute contraindication to donation. Papillary microcarcinoma restricted to the thyroid gland (with no metastases in local lymph nodes) because of its specific behavior and almost always benign course, requires an individualized approach. It seemed that when a recipient is in a life-threatening condition, we should consider taking organs from a donor suffering of papillary microcarcinoma restricted to the thyroid gland.
• living donor transplantation : antiplatelet drugs should be discontinued 2 weeks before; oral contraceptives 6 weeks before; antihypertensive drugs should be reduced; the donor should by hydrated 2 hours before explantation to ensure a diuresis > 80-100 ml/hr; a broad-spectrum antibacterial (2nd or 3rd-generation cephalosporin) is administered and the lower limb are covered with elastic bandage to prevent PE from DVT.
• living related donor (LRD) transplantation / syngenesioplastic transplantation : allogeneic transplantation in which the donor and the recipient have a close biological relationship, such as that of a mother and her child or a brother and sister. With directed donation to loved ones or friends, worries arise about the intense pressure that can be put on people to donate, leading those who are reluctant to do so to feel coerced. In these cases, transplantation programs are typically willing to identify a plausible medical excuse, so that the person can bow out gracefully^ref. Equally important, however, are situations in which people feel compelled to donate regardless of the consequences to themselves. In one instance, both parents of a child who was dying of respiratory failure insisted on donating lobes of their lungs in a desperate but unsuccessful attempt to save her life (Kolata G. Lungs from parents fail to save girl, 9, and doctors assess ethics. New York Times May 20, 1991:A11). Such a sense of compulsion is not unusual. In cases like these, simply obtaining the informed consent of the relative is insufficient — physicians are obligated to prevent people from making potentially life-threatening sacrifices unless the chance of success is proportionately large.
• living nonrelated or unrelated donor (LNRD / LURD) transplantation : allogeneic transplantation in which the donors choose to give to a specific person with whom they have no prior emotional connection. This type of donation usually occurs when a patient advertises for an organ publicly, on television or billboards or over the Internet. Such advertising is not illegal, but it has been strongly discouraged by the transplantation community. 2 central objections are that the practice is unfair and that it threatens the view that an organ is a "gift of life," not a commodity to be bought and sold. Some argue that just as we have a right to donate to the political parties and charities of our choice, so should we be able to choose to whom to give our organs. In practice, however, this means that those who have the most compelling stories and the means to advertise their plight tend to be the ones who get the organs — rather than those most in need. This strikes some ethicists as unfair. Unlike monetary gifts, they argue, organ transplantation requires the involvement of social structures and institutions, such as transplantation teams and hospitals. Hence, the argument goes, these donations are legitimately subject to societal requirements of fairness, and transplantation centers should refuse to permit the allocation of organs on the basis of anything but morally relevant criteria^ref. The most ethically problematic cases are those in which the recipient is chosen on the basis of race, religion, or ethnic group. In one case, for example, the family of a brain-dead Florida man agreed to donate his organs — but insisted that because of the man's racist beliefs, the recipients must be white. Although the organs were allocated accordingly, Florida subsequently passed a law prohibiting patients or families from placing such restrictions on donation (Veatch RM. Transplantation ethics. Washington, D.C.: Georgetown University Press, 2000). Even when the motives for choosing a recipient may be unethical, however, there might be reasons for allowing the donation to proceed. Consider a case that was discussed at a recent public forum hosted by Harvard Medical School's Division of Medical Ethics: a Jewish man in New York learned of a Jewish child in Los Angeles who needed a kidney transplant. The man wanted to help someone of his own faith and decided he was willing to donate a kidney to help this particular child. Despite his discriminatory preference, one might view the donation as permissible, since at least some patients would benefit (the child would receive a kidney, and those below her on the waiting list would move up one notch) and no one would be harmed (those above the girl on the waiting list would not receive the kidney under any circumstances, because the man would not give it to them). Whether directed donation to strangers violates standards of fairness is thus controversial. But if it is permitted, it will be very difficult to prohibit discriminatory preferences, since donors can simply specify that the organ must go to a particular person, without saying why. The other substantial cause for concern about this type of donation is its potential for making possible the buying and selling of organs (see also organ trade). These practices are strictly prohibited by law, yet they seem to be an inherent risk in directed donations to strangers. Wealthy patients in need and healthy donors looking for a quick fix to their financial problems will always be able to find ways around even the most earnest attempts to prevent money from changing hands. Despite these concerns, efforts to direct organ donations to strangers are not new, dating back at least to the celebrated 1982 case of Jamie Fiske, whose father successfully mounted a nationwide appeal for someone to donate a liver to her. Today, many such solicitations are transmitted over the Internet, where, when the practice was relatively limited, organ solicitation was managed quietly, on a case-by-case basis, by individual transplantation centers. All this changed, however, with the emergence in 2004 of MatchingDonors.com (as discussed by Steinbrook in this issue of the Journal, pages 441–444). This Web site currently claims to have > 2100 registered potential donors and to have brokered 12 transplantations, with about 20 more recipient–donor pairs matched and awaiting surgery. Although the business conducted on this organization's Web site does not raise any fundamental ethical issues not already posed by other methods of solicitation, it does introduce a new degree of visibility that increases the magnitude of the issue. Will competing commercial Web sites begin to emerge? How will these sites be held accountable? Dr. Jeremiah Lowney, the medical director of MatchingDonors.com, recently argued that just as a dating service could not be held responsible for a bad date, his Web site has no responsibility for the outcomes of its matches. Furthermore, the Web site has no mechanism for ensuring the quality of the information it provides about transplantation and donation by living persons or for checking the accuracy of information submitted by potential donors and recipients. Given the life-or-death consequences of the procedure, organ donation should not be governed by the ethics of caveat emptor. Nevertheless, MatchingDonors.com has clearly identified a need, and if this need is not met by a service that can address the ethical challenges, the vacuum will be filled by other enterprises. Entrepreneurs commonly open up useful new markets and services that must eventually become subject to rigorous standards and regulations. The solicitation of organs over the Internet is probably here to stay, but it will require higher standards of responsibility and accountability than are currently in place. UNOS has > 20 years of experience in managing the cadaveric-donor pool and is in a good position to extend its jurisdiction to include donation by living donors. The organization recently considered the topic of solicitation and decided not to pursue building a Web site similar to that of MatchingDonors.com but, instead, to provide educational information for anyone who is willing to be a living donor of a kidney and to develop a nationwide mechanism for allocating organs for nondirected donation by living donors. This effort, however, does not go far enough. The proposal does not address directed donation and leaves many critical aspects of donation by living donors to the transplantation centers. Organ transplantation is big business, and each center is highly motivated to expand its share of the pie. They therefore have intolerable conflicts of interest when it comes to regulating themselves. Instead, UNOS should be charged with standardizing the process for evaluating potential donors, ensuring that independent advocates are assigned to help donors make an informed choice, developing mechanisms to deal with potential injury or death to the donor, setting standards for both directed and nondirected donation, and prohibiting transplantation when the chance of success is insufficient to justify the risks. Comprehensive oversight is necessary if the ethical pitfalls are to be adequately addressed^ref
• nondirected donation, in which the donor gives an organ to the general pool to be transplanted into the recipient at the top of the waiting list. The radical altruism that motivates a person to make a potentially life-threatening sacrifice for a stranger calls for careful scrutiny. One recent case involved a man who seemed pathologically obsessed with giving away everything, from his money to his organs, saying that doing so was "as much a necessity as food, water, and air." (Parker I. The gift: Zell Kravinsky gave away millions: but somehow it wasn't enough. The New Yorker. August 2, 2004:54-63). After donating one kidney to a stranger, he wondered how he might give away all his other organs in a dramatic suicide. Other psychologically suspect motivations need to be ruled out as well. Is the person trying to compensate for depression or low self-esteem, seeking media attention, or harboring hopes of becoming involved in the life of the recipient? Transplantation teams have an obligation to assess potential donors in all these dimensions and prohibit donations that arouse serious concern^ref.
Donable tissues : kidney, liver segment II, lung lobe, left pancreas, pancreatic islets
Epidemiology : today, according to the United Network for Organ Sharing (UNOS), almost half of all kidney donors in the USA are living. In 2004, living organ donors also provided a lobe of the liver in approximately 320 cases and a lobe of a lung in approximately 15 cases^ref.
Such grafts are invariably rejected unless the recipient is immunosuppressed, or the graft is to an immune-privileged site.
Click here for pathogenesis of transplant rejection.
Click here for danger theory.
Click here for Italian laws
Criteria for assignation :
• age
• compatibility
• human blood groups
• Rh group doesn't matter
• AB0 group : donor 0 to recipient 0 and B, donor A to recipient A and AB, donor AB to recipient AB, donor B to B and AB
• HLA types (HLA-A, HLA-B, HLA-DR), and MHC typization tools
• waiting time
Common contraindications for donors :
• cancers (breat cancer in females, prostate cancer in males), but nonmelanoma skin cancers is not a contraindication
• sepsis and infection from an antibiotic-resistant pathogen (body cooling impairs detection of many species), but localized infection at other body site is not a contraindication
• viral infections : HBV, HIV-1 patients may receive organs only in pilot programs, HCV patients may donate liver only for fulminating hepatitis, HHV-4 / EBV, HHV-5 / CMV, HHV-3 / VZV should be monitored
• some countries prevent inmates from donating organs
Overall donor risk :
• standard risk
• augmented risk : e.g. episodic finding of drug of abuse
• calculated risk : e.g. time elapsed since cancer remission
• unacceptable : e.g. HIV patients
• xenogeneic transplantation / xenotransplantation / xenotransplant / xenograft / heterograft / heterotransplantation / heterologous or heteroplastic graft (if the donor belongs to a species different from Homo sapiens)

"The dream of the ancients from time immemorial has been the junction of portions of different individuals, not only to counteract disease, but also to combine the potentials of different species. This desire inspired the birth of many mythical creatures which were purported to have capabilities normally beyond the power of a single species. The modern world has inherited these dreams in the form of the sphinx, the mermaid and the chimerical forms of many heraldic beasts." (Christiaan Barnard, 1967)^ref
History : because of the lack of donor organs, the use of biomechanical and nonhuman organs is being explored as a potential solution to this shortage. Xenotransplantation was actually performed long before clinical allotransplantation. In 1905, French surgeon Princeteau transplanted a slice of rabbit kidney into a uremic child. Despite noted improvement of renal function, the child died of pneumonia. Although many other examples of xenografts of different organs have been reported from this early period, human xenografts were quickly abandoned because they uniformly failed. More recently, in 1960, Reemtsma performed a kidney transplantation from a chimpanzee donor into a human recipient; aggressive immunosuppression led to a 9-month survival. In 1984, Bailey performed an orthotopic transplantation of a baboon heart into a human neonate with a hypoplastic left ventricle; the graft functioned for 20 days. Despite aggressive immunosuppression, graft failure occurred secondary to vascular rejection. A baboon-to-human liver transplantation was performed by Starzl in 1993. This graft functioned well for a time, although the patient died several months later from severe sepsis and organ rejection. Other xenotransplant efforts include transplantation of dopaminergic porcine fetal neural cells to the basal ganglia in order to treat Parkinson disease. Deacon et al performed one such transplantation in 1997. Cellular xenografts, such as pancreatic islet cell transplantation and neural cell transplantation, may be protected from rejection because of the absence of vascular tissues. In contrast, xenotransplantation may, in the future, be used as a lifesaving procedure for critically ill patients who are waiting for allografts but are dying because of the shortage of organs. This technique may be particularly helpful for patients with acute liver failure or cardiac failure^{ref1, ref2}
Hurdles to clinical xenotransplantation :
• size and function. Studies in which pig organs have been transplanted into nonhuman primates indicate that the kidneys,hearts, and lungs of pigs would function sufficiently well in a human to sustain life. By contrast, presumed incompatibilities between the complex metabolic systems of the pig and human liver would seem to preclude ready application of hepatic xenotransplantation. However porcine liver xenografts can function adequately in baboons, and pig hepatocytes have been observed to sustain the life of rats with cirrhosis. Even if physiological hurdles were found to be a barrier to xenotransplantation, genetic engineering could be applied to the roblem.
• immunotolerance can be established because the amount of nonspecific immunosuppression required to protect the organs from rejection would be clinically intolerable. The immune responses to xenotransplantation are much more severe than the immune responses to allotransplantation for at least 3 reasons :
• all individuals have innate immunity agains xenogeneic cells, and this innate immune response recruits adaptive immune responses against the graft mediating hyperacute rejection.
• xenogeneic transplants carry a diverse set of foreign antigens against which cellular and humoral immune responses can be elicited (in allotransplants, by contrast, the main foreign antigens are MHC antigens)
• immune regulation, which might partially control responses to allograft, might fail to do so in responses to xenografts
As the biological barriers to xenotransplantation depend, to a significant extent, on the way in which the graft is connected to the recipient, it is importtant to distinguish between
• isolated cell xenotransplants are nourished and maintained by the microenvironment, growth factors and ingrowth of capillaries of the recipient. On the other hand tissue xenotransplants are maintained by both donor and recipient growth factors, and have a mixed vascular supply, consisting of in-grown blood vessels of recipient origin and blood vessels formed by the spontaneous anastomosis of donor and recipient capillaries. In both cases xenotransplant failure may be due to
• primary non-function :
• inability of growth factors of the recipient to support newly implanted cells and/or failure of graft factors to support angiogenesis by host vessels.
• action of NK cells or recently activated T lymphocytes on the newly implanted graft
• action of complement on xenogeneic cells and tissue introduced into the blood (for example, pancreatic islets injected into the portal vein)
• cellular rejection
• whole-organ xenograft are connected to the recipient by anastomosis of large blood vessels of the donor and recipient. As aside from this connetion, the graft remains entirely of donor origin, organ xenografts are not generally compromised by incompatibility of the local environment in which they are placed and xenotransplant failure may be due to
• hyperacute rejection : the a-gal epitope (Gala1-3Galb1-(3)4GlcNAc-R) is abundantly synthesized on glycolipids and glycoproteins of non-primate mammals and New World monkeys by the glycosylation enzyme a1,3galactosyltransferase (a1,3GT). In humans, apes and Old World monkeys, this epitope is absent because the a1,3GT gene was inactivated in ancestral Old World primates. Instead, humans, apes and Old World monkeys produce the anti-Gal natural antibodies (nAb), which specifically interacts with a-gal epitopes and which constitutes approximately 1% of circulating immunoglobulins. Anti-Gal has functioned as an immunological barrier, preventing the transplantation of pig organs into humans, because anti-Gal binds to the a-gal epitopes expressed on pig cells. Binding of xenoreactive nAb to Gala1-3Gal activates complement and rapid insertion of terminal complement complexes in the cell membranes of the endothelial lining of blood vessels in the donor organ, which in turn causes graft destruction^ref. Approaches to prevention of hyperacute rejection include :
• xenoreactive antibodies (XA) can be depleted by column absorption
• complement can be inhibited by Cobra venom factor or soluble CR1 (sCR1), or genetic engineering of donor organs for the expression of the species-specific human complement-regulatory proteins DAF, CD59 / MIRL, and MCP.
• NK cells
• genetic engineering of a-1,3-galactosyltransferase 1 (GGAT1)^-/- pigs to eliminate Gal-a-1,3-Gal epitopes has become possible with cloning technologies. Anyway eliminating an antigen by gene targeting might uncover new epitopes!
• kidneys from GGAT1^-/- pigs enabled 8 baboons to survive for up to 81 days, compared with around 30 days for non-transgenic kidney (the longest that researchers have extended a baboon's life with a pig organ — although baboon-to-baboon transplants are still more successful). Along with the kidney, the researchers also transplanted pieces of pig thymus, which pump out immune cells that do not attack pig tissues.
• acute vascular rejection is caused by xenoreactive antibodies which bind to the xenograft causing activation of endothelium in the graft and possibly apoptosis. Approaches to prevention of acute vascular rejection include :
• pre-transplant infusion with donor haemotopoietic cells induces tolerance to Gal-a-1,3-Gal and other xenospecific antigens
• grafting donor bone marrow or stem cells, but the biological hurdles to engraftement of xenogeneic bone marrow cells, which include the action of antibodies and complement on the cells and the incompatibility of host growth factors, might make induction of tolerance to organ xenografts difficult to achieve.
• grafting donor thymus into initially immunocompetent recipients that are thymectomized and treated with T cell-depleting Abs allows reconstitution of the CD4 compartment, and these donor CD4 cells are rendered tolerant to donor-specific tissue. These hosts appear to have normal immunocompentece, in that they are capable of clearing opportunistic infections and responding to protein Ags presented by recipient class II MHC molecules. The donor thymus is not required to produce more T cells than normal syngeneic thymus to maintain a constant peripheral CD4 cell pool.
• knock-out of a-1,3-galactosyltransferase 1 (GGAT1) and possibly other pig genes to decrease antigen expression has become possible with cloning technologies. Anyway eliminating an antigen by gene targeting might uncover new epitopes !
• suppression of pro-coagulant or pro-inflammatory genes inhibits endothelial cell activation
• accommodation, an acquired resistance to humoral immune-mediated injury, has been induced by transient depletion of xenoreactive antibodies followed by the return of those antibodies without causing humoral rejection. In this setting, accommodation might be brought about by
• a change in xenoreactive antibodies
• a change in the antigens in the graft
• binding of xenoreactive antibodies or the action of inflammatory agonists, in subtoxic amounts induces changes in the graft, which make the graft inured to humoral injury.
• desensitization or loss of receptors for inflammatory agonists
• interruption of cell activation or effector pathways - for example, by IkB or BCL2
• production of proteins, such as CD59 / MIRL or haem oxygenase 1 (HO-1) that repair or block the detrimental effects of the agonists that would otherwise induce tissue injury
• cellular rejection
• chronic rejection : if it is caused by an immune response to the graft, as some experimental evidence indicates, then it should be common and severe in xenotransplants. If it is caused by qualities of the graft, such as preservation time, ischaemia and donor age, then it should not be much of a problem. In any case, because xenotransplantation offers an unlimited supply of organs, the impact of chronic rejection might be less serious as the chronically rejected organ can be replaced
• donor infectious organisms :
• chimpanzees (Pan troglodytes) : theoretically transmittable viruses
• simian immunodeficiency virus (SIV)
• cercopithecine herpes virus 1 / herpes virus B
• simian cytomegalovirus (SCMV)
• simian fomay virus
• monkeypox virus
• pigs (Sus scrofa) : the risk of the spread of pathogens should be less in xenotransplantation than in allotransplantation, because most pathogens of pigs do not infect humans, and because pigs can be raised to be free of known human pathogens. All pigs carry porcine endogenous retrovirus (PERV), but its potential effects on people are unknown and to date it has not been found to be transmitted to humans (strain A uses as receptors HuPAR-1 and HuPAR-2). In a model of human-pig chimerism, some human HSCs engrafted in pigs contain both human and porcine chromosomal DNA. These hybrid cells divide, express human and porcine proteins, and contribute to porcine nonhematopoietic tissues. In addition, the hybrid cells contain porcine endogenous retroviral DNA sequences and are able to transmit this virus to uninfected human cells in vitro. Thus, spontaneous fusion can occur in vivo between the cells of disparate species and in the absence of disease. The ability of these cell hybrids to acquire and transmit retroviral elements together with their ability to integrate into tissues could explain genetic recombination and generation of novel pathogens^ref.
• cows (Bos taurus)
Strategies :
• nuclei from a human patient might be transferred to enucleated stem cells of an animal, and the cells might then be grown in vitro or in an animal to generate differentiated human tissue that is autologous with the patient.
• pig embryonic tissues represent an attractive option for organ transplantation. However, the achievement of optimal organogenesis after transplantation, namely, maximal organ growth and function without teratoma development, represents a major challenge. Distinct gestational time windows were determinated for the growth of pig embryonic liver, pancreas, and lung precursors. Transplantation of embryonic-tissue precursors at various gestational ages [from E (embryonic day) 21 to E100] revealed a unique pattern of growth and differentiation for each embryonic organ. Maximal liver growth and function were achieved at the earliest teratoma-free gestational age (E28), whereas the growth and functional potential of the pancreas gradually increased toward E42 and E56 followed by a marked decline in insulin-secreting capacity at E80 and E100. Development of mature lung tissue containing essential respiratory system elements was observed at a relatively late gestational age (E56). These findings, showing distinct, optimal gestational time windows for transplantation of embryonic pig liver, pancreas, and lung, might explain, in part, the disappointing results in previous transplantation trials and could help enhance the chances for successful implementation of embryonic pig tissue in the treatment of a wide spectrum of human diseases^ref.
Web resources :
• Swine Leukocyte Antigen Database (SLAD)
• Bovine Leukocyte Antigens
Click here for ischemia reperfusion injury (IRI)
Placements :
• homotopic or orthotopic transplantation : transplantation of tissue from a donor into its normal anatomical position in the recipient.
• heterotopic transplantation :
• kidney transplantation
• experimental transplantation of tissue typical of one area to a different recipient site, usually a non-immune-privileged site
• subcutaneous tissue
• subcapsular space of the kidney
• mice given transplanted testicle tissue on to the backs are capable of growing mature goat, pig and monkey sperm 7 months later : this may one day help to preserve endangered species.
Good tissue practices (GTP) rule.
Transplanted material :
• cell transplantations (cell therapy)
• infusion graft : transplantation by injection of a suspension of cells
• adoptive transfer or adoptive immunization => adoptive immunity
• differentiated cells
• CNS neurons
• focal or regionally restricted neurologic diseases
• Parkinson's disease (PD) : the goal has been the engraftment and enhanced survival of dopamine-producing cells within the striatum, or by forestalling degeneration of dopaminergic neurons within the substantia nigra (SN). Anyway differentiated dopaminergic neurons don't survive after transplantation and some are rejected by the immune system. All strategies here reported didn't result in life quality improvement in double blind placebo-controlled trials even if neurons are alive and dopamine producing.
• xenogeneic pig neurons
• dopamine-producing cells from patient's own adrenal glands
• neurons from aborted fetuses (15-50% developed dyskinesias caused by overactive or poorly functioning neurons)
• dopaminergic neurons derived from cloned ES cells by
• transfection with a CMV plasmid driving expression of Nurr1 (a transcriptional factor involved in the differentiation of midbrain precursors into dopamine neurons)
• cell culture system that allows rapid and efficient derivation of most nervous system neural subtypes
• widely disseminated (in the brain and/or spinal cord) neurologic diseases (lysosomal storage diseases (LSD), leukodystrophies, inborn errors of metabolism, hypoxic-ischemic encephalopathy, Alzheimer's disease (AD), Huntington's disease (HD), multi-infarct dementia (MID), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), glioblastomas) : they have not typically been regarded as within the purview of neural transplantation
• transplantation of somatic cells derived from the hematopoietic system administered via bone marrow transplantation (BMT), with unsatisfactory results
• autologous skin cells genetically modified to express NGF when injected stereotaxically in up to 10 million numbers into 10 different brain sites they halve Alzheimer's disease (AD) patients' rate of mental decline 1 year on (by comparison, the best drug therapies offer only around a 5% decrease in the rate of decline) : brain scans also showed that the patients had more blood pumping around their brains than before their surgery, indicating that the injected cells were still alive and having an effect one year later. Skin cells are easy to obtain, culture and alter genetically and replicate when given space to grow in culture but stop dividing when they are implanted in the dense tissue of the brain, meaning that they are unlikely to form tumours : over time the transplanted cells may slow their production of NGF and be less effective
• neonatal pig choroid plexus cells encapsulated in alginate expressing neurotrophins has shown beneficial effects in rat^ref and primate models (225 nmol quinolinic acid injection in the ipsilateral striatum) for Huntington's disease (HD). Living Cell Technologies (LCT)'s (Auckland, New Zealand) BioPharma subsidiary in Providence, Rhode Island, which plans to carry out the human trials.
Glial scarring prevents implantation of transplanted neurons in both brain and retina.
• blood cells : the National and regional register of blood and plasma, established in 1991, makes it possible to know the production and distribution of blood and blood components in Italy. It represents an important instrument to programme the blood needs. The Register is filled out by every Transfusion Service (302 in 2004), according to the standard questionnaire established in the Minister Decree of 1996, it is sent to the relative Regional Health Board and, by this, to the Istituto Superiore di Sanità (the Italian National Institute of Health). The Register is essentially divided into two sections: the first one collects data concerning donors, donations, and plasma; the second one checks other important activities for the transfusion service (laboratory diagnostics, computerisation, quality controls, and committees for the good use of blood). In 2004 total donors were 1,451,641, blood donations were 2,274,513, and donations by aphaeretic procedures about 400,000. The amount of plasma produced was 725,112 litres, 531,395 of them were sent to industry for plasmaderivatives production. About 80,000 units of red cells were exchanged between regions to satisfy the national need^ref.
• RBCs
• transfusion : the introduction of whole blood or blood components directly into the bloodstream
• exchange, exsanguination, replacement or substitution transfusion / exsanguinotransfusion : repetitive withdrawal of small amounts of blood and replacement with donor blood, until a large proportion of the blood volume has been exchanged; used primarily in newborn infants with erythroblastosis fetalis and sometimes in patients with various other blood conditions
• fetomaternal transfusion : transplacental passage of fetal blood into the circulation of the mother; in small amounts it may go unnoticed, but in larger amounts it can cause anemia or edema in the fetus.
• direct or immediate transfusion : the transfer of blood from one person to another without use of an intermediate container or anticoagulant
• mediate or indirect transfusion : transfer of blood from a donor to a flask or other container, and then to the recipient
• intraperitoneal transfusion : infusion of blood into the peritoneal cavity
• intrauterine transfusion : transfusion performed on an unborn infant in utero, often referring to transfusion of Rh-negative blood into the infant's peritoneal cavity in the treatment of erythroblastosis fetalis in utero
• autotransfusion / autologous transfusion : reinfusion of blood or blood products derived from the patient's own circulation; contraindicated in neoplastic patients due to risk of spreading neoplastic emboli.
• intraoperative autotransfusion : the collection, processing, and reinfusion of a patient's blood shed from a wound or body cavity during surgery.
• postoperative autotransfusion : the collection, processing, and reinfusion of the patient's blood shed from the mediastinum following open heart or chest surgery or from the chest following traumatic hemothorax
Donors : every people aged 18-60 and weighing > 50 kg, with SBP = 110-180 and DBP = 50-100, can donate blood. Exclusions may be :
• temporary :
• pregnancy (1 year)
• abortion (1 year)
• menstruating women
• gastric ulcer or duodenal ulcer
• hypersensitivities
• vaccination and/or prophylaxis :
• anti-malaria vaccination (3 years)
• anti-polio vaccination (14 days)
• yellow fever vaccination (14 days)
• drugs :
• NSAIDs (5 days)
• tetracyclines (30 days)
• aspecific immunoglobulins (1 year)
• infectious diseases
• HHV-4 / EBV (1 year)
• rubella virus (3 months)
• HHV-3 / VZV (3 months)
• influenzavirus (7 days)
• Toxoplasma gondii (1 year)
• Brucella melitensis (2 years)
• Rickettsia spp. (1 year)
• major surgery (1 year)
• minor surgery (3 months)
• tattoos (6 months)
• earlobe hole (6 months)
• occasional sexual intercourse (1 year)
• acupuncture (6 months)
• transfusion (5 years)
• permanent exclusion
• drug or alcohol addiction
• epilepsy
• severe diabetes mellitus
• severe systemic arterial hypertension or treatment with b-blockers
• autoimmune diseases
• diffuse dermatoses
• severe rheumatic diseases
• acting or previous hepatitis B or hepatitis C
• neoplasms
• cardiovascular diseases
• severe hematological diseases
• chronic nephropathies
• mental instability
• severe dysendocriniae
Donation of 1 unit (350 ml) of whole blood causes a reduction of 1 g/dl of hemoglobin in the donor after 24 hrs.
• platelets :
• neutrophils : use of recombinant G-CSF to stimulate donors, have made it possible to collect extraordinarily large numbers of normal neutrophils for transfusion into neutropenic patients with life-threatening infections. Because larger doses of neutrophils can be transfused, renewed interest has arisen in the use of neutrophil (granulocyte) transfusions to treat adult oncology patients and progenitor cell transplant recipients, in whom neutropenia complicated by severe infections persists as a significant problem, despite combination antibiotic therapy, recombinant cytokines, myeloid growth factors, and use of mobilized peripheral blood progenitor cells.
• natural killer (NK) cells : purification of human NK cells from leukopheresis products using a clinical-scale 2-step immunomagnetic method :
• CD3⁺ cells are first depleted and then CD56⁺ cells are enriched from the CD3⁺ cell-depleted products. The median percentage of CD3^-CD56⁺ NK cells in the final products is 91.0%, and the median recovery was 48.7%. The median depletion for CD3⁺CD56^- T cells was 5.3 log. Natural cytotoxicity of the purified cells us approximately 5-fold higher than that of unpurified mononuclear cells, and it could be further increased by stimulation of the purified cell with IL-2^ref
Complications :
• immediate reactions
• febrile nonhemolytic transfusion reaction (FNHTR) (1–4:100)
• allergic (1–4:100)
• delayed hemolytic (1:1,000)
• transfusion-related acute lung injury (TRALI) (1:5,000)
• acute hemolytic (1:12,000)
• fatal hemolytic (1:100,000)
• anaphylactic (1:150,000)
• post-transfusion purpura
• acute pulmonary edema, acute renal failure
• transfusion-associated transmission (TAT) of hematogenous infections
• HBV (1:63,000)
• HCV (1:1,600,000)
• HIV-1 (1:1,960,000)
• HIV-2 (none reported)
• HTLV-I and -II (1:641,000)
• malaria (1:4,000,000)
• infectious agents rarely associated with transfusion, theoretically possible or of unknown risk include: HAV, parvovirus B-19, Babesia microti (babesiosis), Borrelia burgdorferi (Lyme disease), Trypanosoma cruzi (Chagas disease), and Treponema pallidum subsp.pallidum, HHV-8 and HGV.
• other complications
• RBC allosensitization (1:100)
• HLA allosensitization (1:10)
• post transfusion graft-versus-host disease (PT-GVHD) / transfusion associated graft versus host disease (TA-GVHD) (rare) : a serious complication of blood transfusion with a high mortality rate. It occurs when immunologically competent lymphocytes from the donor are introduced into an immunoincompetent host who is unable to destroy the donor lymphocytes. The donor lymphocytes engraft, recognize the host as foreign and then attack host tissues. GVHD occurs most commonly after allogenic HSCT and less often after transfusion of non-irradiated cellular blood components especially when the blood donor and recipient share similar HLA antigens^ref. In this case, the recipient does not recognize the transfused donor cells as foreign and cannot reject them. The transfused viable T-cells react against the second haplotype on HLA that are not shared, with the resulting clinical picture of TA-GVHD^ref. Involvement is multi-systemic and patients usually present with high grade fever, a maculopapular erythematous scaly rash, hepatocellular damage with elevated hepatic enzymes, profuse and acute onset diarrhea and hematological manifestation in the form of pancytopenia and its resulting complications^ref. Histological changes are non-specific and primarily affect the skin, liver and the gastro-intestinal tract^ref. Characteristic changes in the skin include epidermal basal cell vacuolation; mononuclear cell infiltration in the epidermis and degeneration of the epidermal basal layer. Bulla formation and ulceration of the skin may also be seen. The liver may show degeneration of the small bile ducts and periportal mononuclear infiltrates associated with hepatocellular and cholangiolar cholestasis. The bone marrow may be hypocellular or aplastic with a lymphocytic or histiocytic infiltration along with haemophagocytosis^ref. Other predisposing factors of GVHD are congenital or acquired immunodeficiency, young age and transfusion with fresh whole blood^{ref1, ref2}. Multi-systemic involvement (bicytopenia, maculopapular erythematous rash, fever, watery diarrhea and jaundice following the blood transfusions with related donor) and the high mortality rate associated with this condition necessitates that pediatricians should be aware of this largely preventable condition^{ref1, ref2}. Clinical signs and symptoms in the initial phase can be confused with a variety of other conditions like Stevens Johnson syndrome, viral infections, drug reactions & reactive and familial erythrophagocytic syndromes. Hence a high index of clinical suspicion and relevant pathological support is required for its diagnosis. TA-GVHD has been reported from India in 3 full term neonates, all of whom died^{ref1, ref2}. Corticosteroids, antithymocyte globulin, cyclosporine and/or growth factors have been recommended for treatment either singly or in various combinations. However, attempts at treatment of TA-GVHD are largely ineffective. Newer modalities that have been tried include nafmostat mesilate, chloroquin and daclizumab. Irradiation of blood products prior to transfusion has reduced if not eradicated transfusion- related GVHD^{ref1, ref2}. Other methods under investigation include photoinactivation with the use of psoralen S59 with UV-A light, or the use of photoactive phenothiazine dyes^ref. The rapid downhill course, poor response to therapy and high mortality associated with transfusion associated graft versus host disease make its prevention a priority. Therefore optimizing blood/component use, reducing inappropriate transfusions and more importantly avoiding the use of related donors for transfusions is a more realistic option of preventing transfusion associated graft-versus-host disease in clinical practice.
Risk prevention : some 60% of global blood supplies go to 18% of the world's people, leaving 82% of the global population inadequately covered. So far, only 40 countries have established a 100% voluntary blood donation system, and < 30% of countries have a nationally coordinated blood transfusion service in place. HIV-contaminated blood still accounts for approximately 5% of HIV infections in Africa. While in many countries more and more testing is being done to make blood safe, the WHO said most developing nations do not test for diseases such as HIV-1 or HBV and HCV. Annually, some 6 million tests that should be done for infections are not done
• presumptive viremic blood donors (PVDs)
• testing of blood donors for HIV-1 and HCV RNA by means of nucleic acid amplification was introduced in the United States as an investigational screening test in mid-1999 to identify donations made during the window period before seroconversion : minipool nucleic acid–amplification testing has helped prevent the transmission of approximately 5 HIV-1 infections and 56 HCV infections annually and has reduced the residual risk of transfusion-transmitted HIV-1 and HCV to approximately 1 in 2 million blood units^ref. The prevalence rates of HBV, HCV, HIV-1, and HTLV infections are lower among tissue donors than in the general population. The prevalence of confirmed positive tests among tissue donors was 0.093% for anti-HIV, 0.229% for HBsAg, 1.091% for anti-HCV, and 0.068% for anti-HTLV. The incidence rates were estimated to be 30.118, 18.325, 12.380, and 5.586 per 100,000 person-years, respectively. The estimated probability of viremia at the time of donation was 1 in 55,000, 1 in 34,000, 1 in 42,000, and 1 in 128,000, respectively. However, the estimated probability of undetected viremia at the time of tissue donation is higher among tissue donors than among first-time blood donors. The addition of nucleic acid–amplification testing to the screening of tissue donors should reduce the risk of these infections among recipients of donated tissues^ref.
• precautionary measure to safeguard the blood supply have been taken in the light of the 1st possible transmission of new variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion, which was reported in December 2003. The transfusion occurred in 1996; the blood donor was well at the time but developed symptoms of vCJD in 1999 and died the following year. The recipient was diagnosed with vCJD in 2003. Since 1997, in view of the uncertainty as to whether vCJD could be transmitted by blood or blood products, the UKBS have put in place a number of other measures to reduce the risk of a potential onward cycle of transmission :
• withdrawal and recall of any blood components, plasma derivatives, or tissues obtained from any individual who later develops vCJD (December 1997);
• importation of plasma from the US for fractionation to manufacture plasma derivatives (announced May 1998, implemented October 1999);
• leucodepletion (removal of white blood cells) of all blood components (announced July 1998, implemented Autumn 1999);
• pmportation of fresh frozen plasma from the United States for patients born on or after 1 Jan 1996 (announced August 2002, to be implemented spring 2004);
• promotion of appropriate use of blood and tissues and alternatives throughout the National Health Service (NHS).
Starting on 5 April 2004, the UK government is to ban people who have received blood transfusions since 1980 from donating blood, based on the premise that BSE did not emerge in Britain until after this date^{ref1, ref2}. The new move will exclude some 52,000 donors, prompting concern that vital bloodstocks will fall : only 6% of the eligible population give blood, so there's a real need to recruit new donors. This is a highly precautionary approach, and the benefit of receiving a blood transfusion when needed far outweighs any possible risk of contracting vCJD. To date there has been only one possible case of vCJD being transmitted by blood, yet the UKBS issue over 2.5 million units of blood every year.
• in the USA between 1986 and 1991 there were 29 deaths related to the bacterial contamination of blood or blood products. This represented about 16% of the transfusion-related fatalities during that time period. 8 of the 29 deaths (1-2 fatalities per year) were related to contaminated red cell products, a risk of 1 death per 9 million red cell transfusions. 7 of the 8 episodes were due to Yersinia enterocolitica. Other Gram-negative bacilli that have been implicated are Serratia spp., Enterobacterspp., and Pseudomonas spp. The source of the organism is thought to be asymptomatic transient bacteremia in the donor. Of note, most of the episodes occurred in blood older than 25 days. It has been demonstrated that at 4°C storage, Yersinia can multiply to high concentrations. Typically, fever and rigors occur within 30' of the transfusion and may rapidly progress to systemic arterial hypotension, disseminated intravascular coagulation, acute renal failure, and death : the organism was isolated from the patient's blood and the red cell transfusion. In a report from New Zealand, the fatality rate was 1 per 104,000 per red cell transfusion, a rate 80 times higher than in the USA. The other 21 deaths were related to platelet transfusions, translating into a fatality rate of 1 per 1 million platelet transfusions. The microbiology of these episodes was more variable, including a variety of Gram-negative bacilli (but not Y. enterocolitica), Staphylococcus spp., and Streptococcus spp.
Click here for Italian laws
Web resources :
• American Association of Blood Banks (AABB)
• American Red Cross
• UK National Blood Service
• UK Dept of Health : Protecting Blood Supplies
• Journals :
• Transfusion Medicine
• Transfusion Science
• bone marrow stromal cells (BMSCs) undergo best engraftment through subcutaneous implantation in a 3D matrix scaffold (collagen sponge, hydroxyapatite particles, hyaluronic acid sponge, ...) rather than intravenous infusion. It is conceivable that the former way not only avoids the loss due to the fact that BMSCs are not normally circulating cells unable to cross the endothelial wall, but also lets the cell create a local microenvironment that enhances cell survival. BMSCs are known to inhibit allogeneic T-cell responses and are therefore being investigated to promote haematopoietic engraftment when co-transplanted with HSCs in BMTs. Previous controversial reports have indicated that TGF-b₁ and HGF might be responsible for this effect. Expression and activity of indoleamine 2,3-dioxygenase (IDO) is upregulated in BMSCs after exposure to low levels of IFN-g, which can be generated by allogeneic T cells. IDO is responsible for the catabolism of tryptophan, which has previously been identified as a T-cell inhibitory effector mechanism. The fact that IDO is not constitutively expressed by BMSCs should enable this inhibitory mechanism to be modulated depending on the therapeutic application^ref.
• muscle-derived stem cells (MDCs) : one of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. The induction of endothelial vessel networks has been reported in engineered skeletal muscle tissue constructs using a 3D multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of VEGF expression in the construct and promoted formation and stabilization of the endothelial vessels. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation of the engineered muscle implants in vivo in 3 different models^ref.
• autologous muscle-derived cells for stress urinary incontinence^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10}
• allogeneic myocytes in Duchenne's muscular distrophy (DMD)
• donor-matched arteries grown in vitro from human muscle cells transfected with hTERT and seed on a hose-shaped scaffold of biodegradable polymer. After 2 months, the support dissolves leaving a dense, muscular, tubular structure. Lining cells are then dropped inside to complete the artificial artery^ref
• allogeneic hepatocytes in the treatment of cirrhosis caused by hepatitis viruses. In such cases, xenogeneic hepatocytes might be preferred over human hepatocytes to avoid reinfection by human viruses.
• dura mater allograft :
• Lyodura^® (B. Braun Melsungen AG, Melsungen, Germany)
• autologous periosteum
• Neuropatch^® (B. Braun Melsungen AG)
• Dacron^® (E.I. du Pont de Nemours and Co., Wilmington, DE)
• Ethisorb^® (Ethicon, Inc., Somerville, NJ).
• Tutoplast^® fascia lata (Tutogen Medical, Inc.)
• Tutoplast^® dura (Tutogen Medical, Inc., Parsippany, NJ). The Tutoplast^® process :
• stage 1 (delipidization) : grafts are bathed in acetone and agitated with ultrasound.
• removes fat, which interferes with healing
• inactivates viruses
• prepares tissue so that subsequent steps penetrate the graft more effectively
• stage 2 (osmotic contrast treatment) : this step is unique to the Tutoplast process, enabling us to remove more antigenicity than any other allograft provider. Grafts receive alternating baths of distilled water and saline. This disrupts the bioburden cell wall integrity and exposes intracellular material for removal or destruction in this step and subsequent steps
• kills bacteria
• destroys and removes unwanted cells
• removes most antigenicity
• further reduces the viral load
• stage 3 (oxidation treatment) : in this step, grafts are bathed in a hydrogen peroxide (H₂O₂) wash.
• destroys remaining proteins
• removes residual antigenicity
• inactivates any remaining viruses exposed during the osmotic bath
• minimizes the potential for graft rejection
• stage 4 (solvent dehydration) : grafts receive a series of 7 acetone baths.
• removes all water from tissue, making graft storable at room temperature for 5 years
• ensures inactivation of any remaining prions or viral agents exposed during the osmotic bath
• preserves the dense collagenous fiber structure of the graft, retaining the original tissue strength
• stage 5 (limited-dose gamma irradiation) : grafts receive low-dose g irradiation (17.8 kGy) in a narrow, rotating sterilization system.
• minimizes irradiation to ensure biomechanical integrity of the allograft1
• eliminates reliance on aseptic process and collection methods
• guarantees sterility following cutting and packaging
• inactivates all remaining viruses
• stem cells

They can can be used to ...
• ... replace cells and cell circuits
• ... create host environments sufficiently rich in trophic and/or neuroprotective support to promote the recovery of damaged (though not died) endogenous cells
• ... practice gene therapy
• ... replace genes, including those coding for ECM molecules (secretion gene therapy for cross-correction of genetic metabolic defects)
• ... deliver antiviral genes (eg IFN-a)
Kinds of stem cells :
• fetal skin cells allotransplantation : the source of the healing cells may prove controversial in countries such as the USA, however, as they came from an aborted fetus. Doctors typically treat deep second- and third-degree burns with skin grafting. In this 2-step surgical procedure a patch of skin is removed from one area of the body and transplanted to cover the wound. This can be effective, but often leaves the patient with a scar and may take months to heal. Fetuses have long been known to have remarkable regenerative abilities, but the cells seemed to confer restorative powers to the burnt skin, allowing the damaged tissue to heal itself. The team doesn't know exactly how the skin cells had this effect. But the technique could work for adult burns, as well as other wounds. The team obtained the fetal cells from a woman whose pregnancy was terminated at 14 weeks. They allowed the cells to divide in the laboratory, and then seeded them onto a bed of collagen - an important protein for skin elasticity - and incubated them for 2 days. This procedure can source several million 100-cm² patches for transplant from a single fetal biopsy. 8 burn victims, ranging in age from 14 months to 9 years, underwent treatment. Tiny swatches of cells were placed onto the burn wounds and covered the area with gauze. The treated wounds took an average of 15 days to heal. Other forms of treating similar burns frequently take up to 6 times as long. The remarkable flexibility of the skin mended with the fetal cells meant that the patients recovered full movement of their hands and fingers. The result not only gave the patients nearly perfect skin, but also spared them the trauma of having a graft taken from elsewhere on their body^ref
• ESCs : there are some roadblocks that currently prevent researchers from putting the cells into patients' bodies :
• immune tolerance : human embryonic stem cells (hESCs) are envisioned to be a major source for cell-based therapies. Efforts to overcome rejection of hESCs include nuclear transfer and collection of hESC banks representing the broadest diversity of major histocompatability complex (MHC) polymorphorisms. Anyway injection of hESCs into immune-competent mice is unable to induce an immune response. Undifferentiated and differentiated hESCs fail to stimulate proliferation of alloreactive primary human T cells and inhibit third-party allogeneic dendritic cell-mediated T-cell proliferation (e.g. after LPS challenge) via cellular mechanisms independent of secreted factors. Upon secondary rechallenge, T cells cocultured with hESCs are still responsive to allogeneic stimulators but fail to proliferate upon re-exposure to hESCs^ref. Expression of CD178 / FasL by ES-like cells allows they to escape immune surveillance, allowing second-set allograft acceptance in experimental models. However, this may not be true of the differentiated tissue derived from the ESCs. The creation of a large pool of ESCs lines would increase the chances of matching MHC antigens. Otherwise immune tolerance can be obtained by :
• eliminating or introducing surface antigens through genetic engineering, but this might lead to genetic instability over time. Interestingly, although compared with somatic cells, ESCs develop a relatively small number of mutations (typically restricted to deletion of a chromosome that is replaced by a second copy of the remaining one) they increase with time in culture.
• cell nuclear replacement (CNR) / nuclear-transfer cloning (i.e. introduction of a nucleus from adult cell into an enucleated oocyte). Anyway some countries ban human cloning without drawing a distinction between cloning for reproductive or for therapeutic purposes, preventing it. It further implies ethical issues about practices to get oocytes (eventually from non-human females) and creation of human embryos for the sole purpose of destroying them to obtain replacement cells for the patient who provided the nuclear DNA.
• how to control the cells' transformations into other types : maintenance of pluripotency in human and mouse ES cells (HESCs and MESCs) can be achieved through functionally reversible activation of Wnt signalling (downregulated upon differentiation) by a pharmacological GSK-3 inhibitor. Although Stat-3 signaling is involved in MESC self-renewal, stimulation of this pathway does not support self-renewal of HESCs
• ES cells cannot be grown without help from mouse feeder cells, which means that they could be contaminated with mouse proteins.
Examples :
• generation of confluent cardiomyocyte monolayers derived from ESCs clone in suspension carrying a construct consisting of the a-cardiac myosin heavy chain (aMHC) promoter driving the neomycin resistance gene was used for antibiotic-driven cardiomyocyte enrichment => cardiac bodies (CB) => seeding of cardiomyocytes with 7 x 10⁴ cell/cm² resulted in a homogeneous monolayer of synchronously contracting cells^ref.
• Id knockout mouse embryos display multiple cardiac defects, but mid-gestation lethality is rescued by the injection of 15 wild-type embryonic stem (ES) cells into mutant blastocysts, allowing some of the offspring to survive for 2 days after birth. Myocardial markers altered in Id mutant cells are restored to normal throughout the chimeric myocardium. Intraperitoneal injection of ES cells into female mice before conception, which travelled across the placenta into the babies, also partially rescues the cardiac phenotype with no incorporation of ES cells. IGF-1, a long-range secreted factor, in combination with WNT5a, a locally secreted factor, likely account for complete reversion of the cardiac phenotype. Thus, ES cells have the potential to reverse congenital defects through Id-dependent local and long-range effects in a mammalian embryo^ref. This implies that doctors could simply inject these proteins, or otherwise mimic their effects with drugs, and would not need to inject stem cells at all, or mothers might boost their levels of IGF-1 by changing their diet or taking a drug. A reduction of just 15-20% of the Id protein impairs the stem cells’ ability to rescue these embryonic mouse heart cells. IGF-1 injected into the mother can cross the placenta and influence fetal cardiac development in the Id knock-out embryo.
• around 300,000 neural stem-cells from 16- to 20-week-old human fetuses cultured with growth-promoting chemicals were transplanted into rat brains 7 days after surgically inducing a stroke. 1 month later, an average of 100,000 of the grafted cells were still alive in the rats' brains and nearly half of them had turned into neurons. The remaining cells still looked like stem cells or had turned into astrocytes, a type of brain cell that supports neurons. Some grafted cells had migrated a millimetre or more away from the injection site towards the damaged part of the brain^ref. The next step is to prove that the cells can reverse paralysis in the rodents, before moving on to primate and human trials. Fetal stem cells have advantages over adult and embryonic alternatives. Adult stem cells do exist in the brain, but they are difficult to obtain, survive less well after transplantation and may be less versatile than their younger counterparts. Hundreds of millions of cells would need to be grown for human clinical trials and it is not clear whether these cells are capable of growth on that scale. Prolonged culture periods can slow cell growth and may give the cells a chance to pick up random genetic abnormalities : another approach would be to add a gene to the stem cells that boosts growth and prevents the formation of genetic defects
• human ESCs transfected with TRAIL gene and injected into mice with brain tumours, home in on the cancer and pump out enough TRAIL to cut the tumour size by an average of 50%, and up to 70% in some cases. The cells are thought to track the tumour by following chemical signals emitted by the immune system molecules that attack, but ultimately fail to destroy, the cancer. Stem cells could be modified to express other anticancer molecules, or a combination of cells, each equipped with different molecules, could be used in concert.
• ESCs are currently the most promising donor cell source for cell-replacement therapy in Parkinson's disease^{ref1, ref2}. A strong neuralizing activity present on the surface of stromal cells, named stromal cell-derived inducing activity (SDIA), has been described. Neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons, have been generated. FGF20, preferentially expressed in the substantia nigra^ref, acts synergistically with FGF2 to increase the number of DA neurons in ES cell-derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms^ref.
• ESCs from mice can help mend the broken hearts of sheep. This cross-species experiment is one more step in finding out whether hESCs can mend the damage done by heart attacks. A heart attack damages the muscle and blood vessels that allow a heart to pump blood around the body. Doctors have long sought a way to repair this damage, and some experts say that ESCs hold the answer. Studies have already shown that ESCs can improve blood flow after an attack in small animals, such as rodents^ref. But in people, ethical controversies have slowed research into the benefits of ESCs for ailing hearts. In human trials scientists have used stem cells from a patient's bone marrow to help the healing. These cells are not as flexible as embryonic ones, but can sometimes be persuaded to turn into the desired cell types. Patients treated this way are able to pump more blood after a heart attack than those who don't get the treatment. But researchers hope that ESCs might have a greater effect. ESCs were tested in sheep to see how well they work in large mammals. Because there is not much call for sheep ESCs, they are hard to source, so the team used mouse cells instead. The researchers injected about 30 million cells into the hearts of 9 sheep that had suffered heart attacks. They found that the sheep pumped blood through their hearts 15% more efficiently than the untreated sheep a month after the injections. Distinctive markers from the stem cells were found inside the sheep hearts, proving that the stem cells had gone to work and replaced some of the damaged tissue^ref. For the moment, the improvement is of the same order of magnitude as that achieved in humans with HSCTs. But the healing should increase over time: other studies have shown the biggest benefit from stem cells 2 months after injections. This form of cardiac therapy holds much future promise. He and his colleagues are currently conducting tests to assess the healing power of hESCs in baboons.
Web resources : World Stem Cell Hub
• olfactory ensheathing glial cells (OECs) : neurosurgeon Huang Hongyun at Chaoyang Hospital Beijing, China, have used fetal tissue transplants to treat more than 450 patients from 2002 to 2004. About 1 to 1.5 million cells are injected per injection site. There is no evidence that injecting more cells would attain better results : an Australian group had done a similar procedure on 3 patients, giving 10 injections and injecting 10 to 15 million cells, but that's a large volume and not good for the spinal cord. No controlled clinical trials have been carried out, although Huang is talking with the Miami Project to Cure Paralysis about designing such studies. In the Chinese Medical Journal in 2003, Huang published a report showing results in 171 spinal cord inury (SCI) patients, 2 to 8 weeks after transplantation. The study used the International Standards for Neurological and Functional Classification of Spinal Cord Injury scale, which gives a best total score of 100 for motor function and 112 for pin-prick (light-touch) sensation. Huang did not report baseline scores, but after OEC transplantation, motor scores increased by 8.3 in patients aged 21 to 30 years and 5.7 in those aged 31 to 40 years. Light-touch scores increased by 15.5 and 12.0 in the same groups^ref. His results represent a credible phase 1 trial that establishes the safety and feasibility of such transplants. Preliminary analyses of the results suggest that the procedure may produce rapid but modest sensory and motor improvements in people from 2 to 40 years after injury
• for amyotrophic lateral sclerosis (ALS), patients, 3 incisions are made, 2 in the frontal lobe (where atrophy is greatest) and the third at the spinal cord around mid-neck, for a total of up to 4.5 million cells
• spinal cord inury (SCI) patients get injection in the spinal cord close to the site of injury, for a total of a total of 1-1.5 million cells
• stroke
• multiple sclerosis (MS)
• cerebral palsy
• brain injuries
• ASCs : since many stem cells are capable of travelling widely within the body and perhaps transdifferentiating into diverse cell types within several organs, there is therefore a need for some mechanism to prevent inappropriate gene expression within these organs (obviously without altering the trafficking and transdifferentiating properties of BMSCs) :
• partial differentiation of BMSCs ex vivo before reintroduction
• identification of a specific BMSC population with a propensity to differentiate into the cell type of choice
• to use tissue-specific promoters
• hematopoietic stem cell (HSC) transplantation (HSCT / HCT)
• mesenchymal stem cells (MSC) are not immunogenic, they do not stimulate alloreactivity, and they escape lysis by CTL and NK-cells. Thus, MSC may be transplantable between HLA-mismatched individuals without the need for host immunosuppression. MSC obtained from human bone marrow have been described as adult stem cells with the ability of extensive self-renewal and clonal expansion, as well as the capacity to differentiate into various tissue types and to modulate the immune system. Some data indicate that leukapheresis products may also contain non-hematopoietic stem cells, as they occur in whole bone marrow transplantation (BMT). However, there is still controversy whether MSC expand in the host after transplantation like blood progenitor cells do. Every sample from total bone marrow transplants exhibited growth of MSC after in vitro culture, but not one of 9 leukapheresis products did. In addition, bone marrow aspirates of 9 patients receiving BMT and of 18 patients after PBSCT were examined for orig