Ossur Scientific E-letter
6 July 2007
Ossur Scientific E-letter Issue #2 2007
Welcome to Ossur Academy's very first scientific e-letter
This issue focuses on tissue engineering, a booming multidisciplinary specialty at the crossroads between life sciences and engineering. An introduction will be followed by recomendations of scientific articles focused on orthopedic applications to help you further explore tissue engineering.
Enjoy the reading!
What is tissue engineering?
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Carnegie Mellon University's Bone Tissue Engineering Initiative |
The field of tissue engineering and the striving of all research in the area can be divided into three components, the cells, the scaffold and the final and ultimate product: the tissue. All of them create critical challenges.
Cells
When developing tissue, cells must first be harvested, isolated and expanded in a specific tissue culture. One major problem has been to maintain proliferation (multiplying cells in the process of reproducing new cells) until an adequate amount of cells is available, since the cells will age with a lesser ability to proliferate. Attempts have been made to keep up the cell proliferation by developing recipes, which include growth factors. The same problem has also been counteracted by using adult stem cells.
Adult stem cells are undifferentiated cells found among differentiated cells in fully formed tissues or organs (compared to embryonic stem cells that are derived from a fetal source). Unlike embryonic stem cells, adult stem cells are pluripotent, which means they are able to differentiate into many cell types. The embryonic stem cells, that can differentiate into all cell types, are totipotent. For developing new cells, adult stem cells can proliferate into specialized cells to constitute particular tissues or organs. Sources for adult pluripotent stem cells could be muscle, bone marrow, brain and fat tissue.
Scaffolds
The next critical component of tissue engineering is the scaffold. The scaffold is a three dimensional framework for cells to attach, proliferate and deposit extracellular matrix. The scaffold could be manufactued by the CAD/CAM technique. Ideally the scaffold should be biocompatible, have structural integrity and also act as a temporary carrier for the cells until the new tissue can regenerate and be implanted into the body. The scaffolds normally mimick the structure of the tissue to be produced.
The characteristics of the scaffolds must combine immunologic compatibility, sufficient mechanical strenghth and biodegradability. It is suggested that tissue under construction needs different types of load to optimize them for their future work; blood vessel constructs will need fluid forces, cartilage constructs will need compressive force and tendon constructs will need strain force and shear stress.
Tissue
The next step is to culture and implant the scaffolds to induce and direct the growth of new, healthy tissue to restore structure and function. In tissues like bone, the structure is the function. In more complex tissues, like muscles, complex functions have to be recreated.
Skin, cartilage, bone, vasculature, heart, breast, kidneys and liver are examples of tissue engineered substitutes that are currently being developed throughout the world.
Engineered tissues in the orthopaedic field
Nerves
Nerve reconstruction is a unique challenge because the effects of nerve injury are not situated at the same place as the nerve injury. Autologous (derived from the patient’s own body) nerve grafting includes substitutes such as vessels, muscles or tendon. The benefits are a minimal risk of triggering the immune response. The disadvantages are differences in the structural properties and an increased risk of scar tissue and fibrosis.
By using biodegradable synthetic substitutes to replace nerves the risk of a late foreign-body reaction has decreased. Studies showed a superior sensory recovery compared to autografts in gaps less than 3cm. Today it is not clear if those findings will hold up for longer defects and for larger mixed nerves. In nerve regeneration it is essential to have Schwann cells surrounding the axons. In experimental models it has been found that scaffolds seeded with Schwann cells has improved the regeneration process. Unfortunately there have been problems in culturing, expanding and harvesting those cells, but recently there has been an interest in using bone marrow stem cells to differate, which is interesting, since it is possible to harvest a large amount of cells from a bone-marrow aspiration or even from a vein puncture. Probably newer conduits will also include elements of extracellular matrix, with for example collagen and growth factors.
Today, there are synthetic acellular conduits used clinically, other advances in tissue engineering of nerves are still in the experimental stage. The step from the laboratory bench to bedside has to be preceded by convincing outcome evidence.
Bones
Bone has a regenerative ability, but when the loss of bone tissue is great or the microenvironment is unfavourable there is a need for bone grafting. Since the available amount of bone for grafting is limited and complications from harvesting grafts exists, engineering of bone tissue is an area of high research activity. Several alternatives of donor cells are available, all with different benefits and disadvantages. Osteoblasts (=cells from which bone develop) heve the benefits of their matching but the number of cells from a biopsy are low and they also age early. Fat derived stem cells have shown a high bone formation in vivo. Mesenchymal stem cells from bone marrow are attractive since they have a high proliferation rate and the possibility into differentiate to bone cells. These mesenchymal stem cells are a candidate for “off-the-shelf” bone substitute, thanks to their low immunogenic ability.
Growth factors give important signals for the donor cells to differentiate into osteoblasts and also influence other steps in the repair process. A study of the repair of tibial gaps has shown positive results.
The chosen materials for scaffolds are biodegradable polymers, because they provide initial structural support and degrade without toxic metabolites when the new bone has been developed. The most popular synthetic material has a long history of use as suture material. One disadvantage of these materials is that they are hydrophobic and because of that do not provide the ideal environment for cell material interactions. Research is continuing the properties of the parameters of scaffolds to provide cell distribution, adherence, differention and bone formation.
Bone has today been substituted in three patients with 4-7cm diaphyseal defects. All showed good callus formation and bone integration with a return of function. The patients needed external fixation between 6 and 13 months. Further clinical assessments are still required.
Tendon
Devastating tendon injuries are a major problem, since there is a lack of donor tendons and they easily develop. Engineered tendons have been constructed in several ways to solve this problem. Fibroblasts from tendons or skin and mesenchymal stem cells from bone marrow are examples of donor cells. A study has shown that a mesenchymal seeded scaffold, to bridge a rabbit’s achilles tendon, had better histologic and biomechanical properties than a scaffold alone. Another study on rabbits showed that fibroblast contracted collagen gel could effectively reconstruct defects of the patellar tendon. Tendon reconstructions have also been performed by using biodegradable synthetic constructions. A new strategy, without a scaffold, is to let tendon fibroblasts secrete and assemble their own matrix.
Today most research is focused on extrasynovial reconstructs, more effort has to be placed in the intersynovial tendon reconstructs with properties similar to native tendons.
Skin
Skin substitutes are the most developed engineered tissue and are also currently available for clinical use. In vitro cultured skin substitute from clonal keratinocytes applied to burns resulted in healing. This method has drawbacks such as a long production time, high cost and the result is a fragile, prone to infections and a cosmetically poor skin. The poor outcome is due to the absence of basement membrane and dermis. The stability of the skin, along with other factors, is dependant on these two important structures. Therefore various scaffolds have been used in skin substitutes. These substitutes could be cadaveric skin, skin substitutes based on collagen or hyaluron and synthetic polymers. Combinations of synthetic and biological materials have also been used. Some of those materials are acellular and can be used as a rapid wound cover and a base for subsequent grafting or cell culture. Good clinical results have been achieved by those.
Bilayered dermal substitute products are constructed by dermal cellular elements covered with epithelial cellular elements with collagen lattice, these have have been applied on venous ulcers and surgical wounds.
An ideal skin substitute should hold the same physical characteristics and functions as normal skin, which includes barrier function. Evidence has shown that treatment with artificial skin substitutes, in combination with compression bandages resulted in better healing rate, than treatment with simple dressings. That review also found that further rigorous studies need to be done.
Vessel
There is a great need for vascular graft material for coronal artery and limb vascular reconstruction. Today synthetic grafts are used routinely for reconstruction of large vessels, but suffer from disadvantages such as a high rate of thromboses in thinner vessels. Autografts have less problems with thrombosis and also have a better compliance than synthetic replacements.
The endothelium is found to be important in avoiding thombosis, since it contains antithrombotic and anticoagulant functions, as well as resistance to intimal hyperplacia. The earliest efforts of engineered vessels was synthetic scaffolds seeded with endothelial cells.
Other cell types, like smooth muscle cells and fibroblasts, have been used to create other layers of the vessel. They give the construct extracellular matrix production, structural organisation and an ability to contract. The problems with those constructs includes thrombosis in thin vessels, the nonbiodegradable scaffolds prevents normal remodeling and the synthetic scaffolds can not grow, which causes problem in pediatric surgery. Successful attempts to solve these problems has been to use collagen or synthetic reinforcement and to stimulate the orientation of the smooth mucle cells by applying a pulstative flow or a magnetic field. The biomechaniqal stress effects the cell orientation and extracellular matrix production.
It is very time consuming to produce a vessel construct, 8 weeks are required for adequate cell proliferation and maturation.
There is still limited clinical experience with of tissue engineered vessels. Reconstructions of lower limb vessels, consisting of an endothelial cell lined synthetic graft, has shown a better rate of maintaining circulation than unseeded grafts.
Despite successful attempts of vascular reconstructions challenging problems remain, such as thrombogenesis and biomechanical properties persists until the ideal solutions are found.
Cartilage
Cartilage has a limited intrinsic ability to heal. If it heals the healing tissue has less favourable properties than uninjured cartilage, which causes problems such as pain and limited function of joints. Tissue engineered substitutes are therefore needed.
Today tissue engineered cartilage is already used clinically in the knee joint. The technique is based on an ex vivo expansion (20 to 50 fold) of chondrocytes taken from the joint. The cells are then implanted in the area with the defect. This technique is best for limited areas of destruction and not in combination with ligament or menicus injuries. A study reported good or excellent outcome after that type of treatment in 51 of 61 patients at their 11-year evaluations. Bone marrow mesenchymal stem cells also have the ability to differentiate into chondrocytes and are an attractive alternative.
The biomechanical properties of the scaffold are extremely important since cartilage experiences high compressive and shear stress. Synthetic and biologic materials that have been used for bone tissue engineering can also be used for cartilage substitutes. A new approach has been to inject the scaffold, which contains fibrin, alginate and synthetic hydrogels. Apart from being able to be administered in a minimally invasive manner, they can also incorporate cells and fill defects that are irregulary shaped.
Even though engineered cartilage tissue has been applied clinically, it has still not achieved the same levels of function as native cartilage. One reason could be that the substitutes do not take into account the normal zonal differences in anatomy and physiology. Newer constructs aim to address that problem.
Composite Tissue
The experience of developing composite tissues has been limited. Engineered phalanges with joints have been constructed using biodegradable scaffolds seeded with cells and preshaped to resemble phalanges. These were implanted in mice, tests showed new bone formation, mature hyaline cartilage and a joint capsule at the joint. By using different biomaterials the requirements for differerent tissues were determined.
One clinical use of tissue engineered composites has been reported. It was a reconstruction of a thumb with a interphalangeal joint after amputation. The composite was made of a coral-based scaffold seeded with expanded autologous periosteal cells. The thumb had a normal length and the patient was able to perform most daily activities after 28 months. Improvements of the technique are still needed before routine clinical use can be considered.
Is tissue engineering a reality or science fiction?
Tissue engineering has advanced rapidly in a short time span. The technique is on the doorstep of producing clinical benefit. Today skin and bone substitutes are available and are continually improving. Skin grafts are used in burn reconstruction or in certain inherited skin disorders. Bone substitutes have applications in fracture bone defects and after bone tumour reconstruction. Nerve substitutes have been successfully applied in digital nerve reconstruction and the use will be extended to reconstruction of longer defects and in larger, diverse nerves.
Later, other tissue substitutes will appear. Today at present, the tendon, cartilage and vascular substitutes have poor biomechanical properties and need further development. Once these obstacles are overcome; tendon constructs are likely to use autologous cells in a scaffold; cartilage substitutes will be used in joints where motion is critical. It is expected that vascular substitutes will be used in larger vessels, such as for arterial reconstruction.
Other obstacles for the development of engineered tissue include safety issues and ethical considerations. Controlled clinical studies are one very important tool to assess the safety of a treatment. Ethical consideration is currently, being discussed and an awareness among scientists, as well as biotechnology companies exists.
The holy grail of tissue engineering is to achieve satisfying results with the development of composite tissues. These are materials that would be used when multiple tissues have been lost or destroyed.
Sources
Chong K S A. Chang J. Tissue Engineering for the Hand Surgeon:
A Clinical Perspective The Journal of Hand Surgery / Vol. 31A No. 3 March 2006
Tissue engineering network. 20070509 http://www.tissueeng.net/
Presentation of selected articles
Bellamkonda R V. Peripheral nerve regeneration: An opinion on channels, scaffolds and anisotropy. Biomaterials 27 (2006) 3515–351
Bellamkonda reports about the options of repairing peripheral nerve gaps. The current use of autografts (tissue from the patient's own body) has many disadvantages. Therefore many attempts have been performed to substitute that technique. This report shows that, as yet no definitive engineered alternative has been identified, but several promising methods are forthcoming.
The constructs, which are most likely to succeed, are inspired by the understanding of the distribution and structural and biochemical features of autografts. The author gives an example of design of constructs that mimic autografts’ anisotropic physical features with orientation of columns of Schwann cells and laminin-1, in combination with biochemical features such as anisotrophically distributed trophic factors and/or extracellular matrix elements.
Bellamkonda also highlights the importance of evaluate the engineered constructs suitability by using appropriate animal models and outcome measure methods.
Chong K S A. Chang J. Tissue Engineering for the Hand Surgeon:
A Clinical Perspective The Journal of Hand Surgery / Vol. 31A No. 3 March 2006
Chong and Chang have reviewed the principles and current research of engineered musculoskeletal tissue and its applications in clinical hand surgery.
The authors highlight the current state of tissue engineering of these needed tissues.
Giannoudis P. Pountos I. Tissue regeneration The past, the present and the future. Injury, Int. J. Care Injured (2005) 36S, S2—S5
Giannoudis and Pountos describe how tissues have been, are and might be regenerated. They present the span from amputation, as the only solution for large injuries to bone grafting, distraction technique, endoprosthetic arthroplasty and substitutes for destroyed cartilage, to the use of stem cells.
HAIRFIELD-STEIN M. ENGLAND C PAEK H J. GILBRAITH K B. DENNIS R. BOLAND E. KOSNIK P. Development of Self-Assembled, Tissue-Engineered Ligament from Bone Marrow Stromal Cells. TISSUE ENGINEERING Volume 13, Number 4, 2007
Hairfield and colleagues reports about their engineered scaffold free ligament, made of bone marrow stromal cells from pigs, seeded on laminin-coated substrates with silk suture segments. The cells developed and organised themself in a rod like tissue. Mechanically and histologically the tissue resembled native embryonic connective tissue The tissue were tensile stressed and tolerated an ultimate stress of approximately 15% of native adult mouse tissue.
Rodriguez F J. Valero-Cabre A. Navarro X. Regeneration and functional recovery following peripheral nerve injury. Drug Discovery Today: Disease Models Vol. 1, No. 2 2004
In this article Navarro and colleagues report about the regenerative response of the experimental efforts to heal damaged peripheral nerves. The studied experiments include nerve repair, drug delivery and in vivo tissue engineering.
Stylios G. Wan T. Giannoudis P. Present status and future potential of enhancing bone healing using nanotechnology. Injury, Int. J. Care Injured (2007) 38S1, S63—S74
This article is an overview of the state of materials used for engineered bone tissue today. Porous implant substrates have been the result of a variety of fabrication processes that can be a help in unsolved clinical problems. The materials have been evaluated for their mechanical and biomedical properties and performance to best substitute bone tissue. The authors conclude that a scaffold should be biocompatible with non-toxic degradation products that could be easily excreted and they should act as a 3D template for bone growth both in vitro and in vivo. To do that, they have to consist of a porous network to allow body fluid transport through the pores, this will trigger bone ingrowth, cell migration, tissue ingrowth, and finally vascularisation
Sun W. Starly B. Darling N A. Bio-CAD modeling and its applications in computer-aided tissue engineering. Computer-Aided Design 37 (2005) 1097–1114
Sun et al gives an overview of the advances of the Bio-CAD technique and its applications, such as modeling, design, analysis, simulation and fabrication. They present the methodology and software to create 3D models from images and the generation of blueprint modeling as well as the use of bio-CAD model for the description and representation of tissue physiological properties.
Woo s L Y. Abramowitch S D. Kilger R. Liang R. Biomechanics of knee ligaments: injury, healing, and repair. Journal of Biomechanics Volume 39, Issue 1, 2006, Pages 1-20
This paper reviews the current state of the biologics and biomechanics of the ligaments of the knee and the healing process after injury. The authors approach the technique of tissue engineering with healing and reconstruction of destroyed knee ligaments.
Conclusion
We can conclude that our human body has the ability to recreate damaged tissues, as many animals and insects can. We though need help from scientists. When tissue engineering is mastered, new families of materials, beside drugs, medical devices and tissue- or organ-transplantions will be offered to patients that will improve their lives.
Vision for the P&O field
With the aim of increasing bony length, the Ilizarov technique has been used to improve the cosmetic and functional condition for patients with disabilities. Tissue engineering can offer improved function for the amputee with short residual limbs.
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