Bridging the regeneration gap: stem cells, biomaterials and clinical translation in bone tissue engineering

Advances in our understanding of skeletal stem cells and their role in bone development and repair, offer the potential to open new frontiers in bone regeneration. Tissue engineering seeks to harness the regenerative capacity innate to bone for the replacement of tissue lost or damaged through a broad range of conditions associated with an increasingly aged population. The strategy entails ex vivo expansion of multipotential populations followed by delivery to the site of damage on dynamically durable-biodegradable three-dimensional structures which provide the requisite extracellular microenvironment for stem cell driven tissue development. This review will examine bone stem cell biology, and current advances in skeletal tissue engineering through the enhancement and marrying of biologically informed and clinically relevant strategies.     

Replating of bioreactor expanded human bone marrow results in extended growth of primitive and mature cells

The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC₈ (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC₅ in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC₈. The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC₅ expansion has now been extended to LTC-IC₈ expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation.     

The Chemical Uncoupler 2,4-Dinitrophenol (DNP) Protects against Diet-induced Obesity and Improves Energy Homeostasis in Mice at Thermoneutrality

The chemical uncoupler 2,4-dinitrophenol (DNP) was an effective and widely used weight loss drug in the early 1930s. However, the physiology of DNP has not been studied in detail because toxicity, including hyperthermia and death, reduced interest in the clinical use of chemical uncouplers. To investigate DNP action, mice fed a high fat diet and housed at 30 °C (to minimize facultative thermogenesis) were treated with 800 mg/liter DNP in drinking water. DNP treatment increased energy expenditure by ∼17%, but did not change food intake. DNP-treated mice weighed 26% less than controls after 2 months of treatment due to decreased fat mass, without a change in lean mass. DNP improved glucose tolerance and reduced hepatic steatosis without observed toxicity. DNP treatment also reduced circulating T3 and T4 levels, Ucp1 expression, and brown adipose tissue activity, demonstrating that DNP-mediated heat generation substituted for brown adipose tissue thermogenesis. At 22 °C, a typical vivarium temperature that is below thermoneutrality, DNP treatment had no effect on body weight, adiposity, or glucose homeostasis. Thus, environmental temperature should be considered when assessing an anti-obesity drug in mice, particularly agents acting on energy expenditure. Furthermore, the beneficial effects of DNP suggest that chemical uncouplers deserve further investigation for the treatment of obesity and its comorbidities.     

Optimization and mathematical modeling of the transtubular bioreactor for the production of monoclonal antibodies from a hybridoma cell line

This report describes the use of a transtubular bioreactor to study the relative effects of diffusion versus perfusion of medium on antibody production by a hybridoma cell line. The study was performed with a high-density cell culture maintained in a serum-free, low-protein medium for 77 days. It was determined that the reactor possessed a macro-mixing pattern residence time distribution similar to a continuous stirred tank reactor (CSTR). However, due to the arrangement of the medium lines in the reactor, the flow patterns for nutrient distribution consist of largely independent medium path lengths ranging from short to long. When operated with cyclic, reversing, transtubular medium flow, some regions of the reactor (with short residence times) are more accessible to medium than others (with long residence times). From this standpoint, the reactor can be divided into three regions: a captive volume, which consists of medium primarily delivered via diffusion; a lapped volume, which provides nutrients through unilateral convection; and a swept volume, which operates through bilateral convection. The relative sizes of these three volumes were modified experimentally by changing the period over which the direction of medium flow was reversed from 15 min (larger captive volume) to 9 h (larger swept volume). The results suggest that antibody concentration increases as the size of the diffusion-limited (captive) volume is increased to a maximum at around 30 min with a sharp decrease thereafter. As reflected by changes in measured consumption of glucose and production of lactate, no significant difference in cellular metabolism occurred as the reactor was moved between these different states. These results indicate that the mode of operation of the transtubular bioreactor may influence antibody productivity under serum-free, low-protein conditions with minimal effects on cellular metabolism.     

Postmortem Changes in Binding to the Muscarinic Receptor from Human Cerebral Cortex

Abstract: The effects of storage at 4°C on the antagonist and agonist binding properties of the muscarinic acetyl-choline receptor from fresh surgical and frozen autopsy samples from human cerebral cortex were studied. The number of 1-[³H]3-quinuclidinyl benzilate binding sites and their affinities were stable up to 51 h, both when stored as pieces of intact nonfrozen tissue and as a homogenate. The agonist binding properties as measured by the ability of the muscarinic agonist carbachol to compete with l-[³H]3-quinuclidinyl benzilate were also stable up to 51 h when the tissue was stored in the form of pieces. The affinity for carbachol decreased when the tissue was stored as a homogenate. The frozen autopsy samples showed no significant differences in binding properties in comparison with fresh neurosurgical tissue.     

Callus induction and regeneration in<Emphasis Type="Italic"> Spirodela</Emphasis> and<Emphasis Type="Italic"> Lemna</Emphasis>

The development of tissue culture systems in duckweeds has, to date, been limited to species of the genus Lemna. We report here the establishment of an efficient tissue culture cycle (callus induction, callus growth and plant regeneration) for Spirodela oligorrhiza Hegelm SP, Spirodela punctata 8717 and Lemna gibba var. Hurfeish. Significant differences were found among the three duckweed species pertaining to carbohydrate and phytohormone requirements for callus induction, callus growth and frond regeneration. In vitro incubation with poorly assimilated carbohydrates such as galactose (S. oligorrhiza SP and L. gibba var. Hurfeish) and sorbitol (S. punctata 8717) as sole carbon source yielded high levels of callus induction on phytohormone-supplemented medium. Sorbitol is required for optimal callus growth of S. oligorrhiza SP and S. punctata 8717, while sucrose is required for callus growth of L. gibba var. Hurfeish. Sucrose either alone (S. oligorrhiza SP, L. gibba var. Hurfeish) or in addition to sorbitol (S. punctata 8717) is required for frond regeneration.Abbreviations ABA: (±)-Abscisic acid - BA: N⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - Dicamba: 3,6-Dichloro-2-methoxybenzoic acid - 2iP: N⁶-(2-Isopentenyl)adenine - NAA: -Naphthaleneacetic acid - PCA: p-Chlorophenoxy acetic acid - Picloram: 4-Amino-3,5,6-trichloropicolinic acid - TDZ: Thidiazuron Communicated by A. AltmanJ. Li and M. Jain contributed equally to the research reported in this article.     

Effects of feeding regime on growth rate in the Mediterranean Sea anemone Actinia equina (Linnaeus)

Polyps of Actinia equina are the most common sea anemones in the rocky intertidal zone of the Mediterranean coast of Israel, where they occur in one of the southernmost populations of this species in the northern hemisphere. We examined effects of feeding rate on polyp growth at ambient sea temperature for this population. Under laboratory conditions, polyps were left unfed, or were fed with brine shrimp (Artemia) once every 2 weeks, once a week, or twice a week. Of the four experimental treatments, only feeding twice a week resulted in polyp growth; under all other regimes, the sea anemones lost body mass. We conclude that a high rate of feeding is required at sea temperatures in the eastern Mediterranean, where these sea anemones may have high metabolic rates relative to more northern populations.     

基因型和胚龄对小麦未成熟胚离体培养反应的影响

本文对34种基因型的小麦未成熟胚在离体培养中的反应进行了比较。结果表明,94％的供试基因型愈伤组织诱导率都可达到80％以上,若排除供体植株环境条件的不同和接种过程中的人为因素可能造成的影响,不同基因型的愈伤组织诱导率看来没有根本的差异。愈伤组织分化率因基因型的不同变动在0—60％之间,平均为32.7％。虽然同一基因型的盾片愈伤组织分化率在不同年份中有所不同,但是愈伤组织是否具有再生能力?看来是个稳定的遗传性状。因此小麦未成熟胚对愈伤组织诱导的反应和愈伤组织的再生能力可能具有不同的遗传基础。本文的结果还表明,虽然最适于培养的未成熟胚的大小为1毫米左右,伹小至0.3毫米的未成熱胚仍能以几乎100％的频率形成愈伤组织,60％左右的愈伤组织能分化出再生檀株,只是所需的时间比1毫米左右的胚较长。     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.